USO09598422B2
(12) United States Patent (10) Patent No.: US 9,598.422 B2 Beck et al. (45) Date of Patent: Mar. 21, 2017
(54) IMMUNOREGULATORY AGENTS A6 IK3I/445 (2013.01); A61K 31/4412 (71) Applicant: Flexus Biosciences, Inc., Princeton, NJ (2013.01); A61K 31/47 (2013.01); A61 K 3 1/4709 (2013.01); A61K 31/496 (2013.01); (US) A6IK3I/519 (2013.01); A61 K3I/5377 (72) Inventors: Hilary Plake Beck, Emerald Hills, CA (2013.01); A61K 45/06 (2013.01); C07C (US); Juan Carlos Jaen, Burlingame, 2II/45 (2013.01); C07C 233/65 (2013.01); CA (US); Maksim Osipov, Belmont, C07C 255/50 (2013.01); C07D 2II/08 CA (US); Jay Patrick Powers, Pacifica, CA (US); Maureen Kay Reilly, (2013.01); C07D 213/68 (2013.01); C07D Belmont, CA (US); Hunter Paul 215/04 (2013.01); C07D 215/233 (2013.01); Shunatona, San Francisco, CA (US); C07C 2101/14 (2013.01) James Ross Walker, Menlo Park, CA (58) Field of Classification Search (US); Mikhail Zibinsky, Lodi, CA None (US); James Aaron Balog, See application file for complete search history. Lambertville, NJ (US); David K. Williams, Delran, NJ (US); Jay A. (56) References Cited Markwalder, Lahaska, PA (US); Steven P. Seitz, Swarthmore, PA (US); U.S. PATENT DOCUMENTS Emily Charlotte Cherney, Newtown, 5,455,273 A 10, 1995 Maier et al. PA (US); Liping Zhang, East Windsor, 5,723,464 A 3/1998 Brightwell et al. NJ (US); Weifang Shan, Princeton, NJ 7,645,771 B2 1/2010 Kazmierski et al. (US); Weiwei Guo, Lawrenceville, NJ 8,088,803 B2 1/2012 Combs et al. (US); Audris Huang, New Hope, PA 2002fOO16463 A1 2/2002 Zablocki et al. 2003. O190298 A1 10/2003 Bradley et al. (US) 2004.0029887 A1 2/2004 Bhatia et al. (73) Assignee: Flexus Biosciences, Inc., Princeton, NJ 2004/0234623 A1 11/2004 Munn et al. (US) 2006/0258719 A1 11/2006 Combs et al. 2007/0129347 A1 6/2007 Hinze et al. (*) Notice: Subject to any disclaimer, the term of this 2007/O197584 A1 8, 2007 Schwink et al. patent is extended or adjusted under 35 2008.003.9453 A1 2/2008 Putman U.S.C. 154(b) by 0 days. 2008O146569 A1 6/2008 Blake et al. 2009/0275523 A1 11/2009 Schudok et al. (21) Appl. No.: 14/933,879 2010, 0008866 A1 1/2010 Blum et al. 2010. 0233166 A1 9/2010 Prendergast et al. (22) Filed: Nov. 5, 2015 2011/0218183 A1* 9, 2011 Chen ...... CO7D 253/O7 (65) Prior Publication Data 514,210.18 2011/0306644 A1 12/2011 Hoekstra et al. US 2016/O137653 A1 May 19, 2016 2013/O197095 A1 8, 2013 Nolte et al. 2013/02177O6 A1 8, 2013 Tran et al. Related U.S. Application Data 2014/0212444 A1 7/2014 Holoshitz et al. (60) Provisional application No. 62/075,678, filed on Nov. 2016.0137652 A1 5, 2016 Becket al. 5, 2014. 2016.0137653 A1 5, 2016 Becket al. (51) Int. Cl. FOREIGN PATENT DOCUMENTS C07C 255/50 (2006.01) A 6LX 3L/277 (2006.01) EP O596.298 B1 1, 2002 CD7C 233/65 (2006.01) EP 1781656 B1 12/2007 CO7D 487/04 (2006.01) (Continued) A6 IK3I/I66 (2006.01) CO7D 2II/08 (2006.01) OTHER PUBLICATIONS A6 IK 3/445 (2006.01) CD7C 2 II/245 (2006.01) Robinson et al. Tetrahedron (2003), 14(1): p. 1407-1446.* A6 IK3I/36 (2006.01) (Continued) CO7D 25/04 (2006.01) A6 IK 3/47 (2006.01) Primary Examiner — Yong Chu A 6LX 3L/24709 (2006.01) (74) Attorney, Agent, or Firm — Baker & Hostetler LLP C07D 215/233 (2006.01) CO7D 213/68 (2006.01) (57) ABSTRACT A6 IK 3/442 (2006.01) Compounds that modulate the oxidoreductase enzyme A6 IK 3/496 (2006.01) indoleamine 2,3-dioxygenase, and compositions containing A 6LX 3/59 (2006.01) the compounds, are described herein. The use of such A 6LX 3/5.377 (2006.01) compounds and compositions for the treatment and/or pre A6 IK 45/06 (2006.01) vention of a diverse array of diseases, disorders and condi A6 IK3I/00 (2006.01) tions, including cancer- and immune-related disorders, that (52) U.S. Cl. are mediated by indoleamine 2,3-dioxygenase is also pro CPC ...... C07D 487/04 (2013.01); A61K3I/00 vided. (2013.01); A61K 31/136 (2013.01); A61 K 3 1/166 (2013.01); A61 K3I/277 (2013.01); 34 Claims, 4 Drawing Sheets US 9,598.422 B2 Page 2
(56) References Cited Munsen et al., Ligand a versatile computerized approach for char acterization of ligand-binding systems, Analytical Biochemistry, 107(1), 220-239, Sep. 1980. FOREIGN PATENT DOCUMENTS Platten, M. et al., Tryptophan catabolism in cancer: beyond IDO and WO WO 99,2931.0 6, 1999 tryptophan depletion, Cancer Research, 72(21):5435-5440, Nov. WO WOOO, 56727 9, 2000 2012. WO WO O1/92204 12/2001 Ishiyama, et al., Palladium (0)-catalyzed cross-coupling reaction of WO WO 2004/094409 11 2004 alkoxydiboron with halorenes: a direct procedure for arylboronic WO WO 2005/08O317 9, 2005 esters. J. Org. Chem, 60, 7508-7510, Nov. 1995. WO WO 2006/O18279 2, 2006 WO WO 2006/02987.9 3, 2006 El-Faham, et al., Peptide coupling reagents, more than a letter Soup, WO WO 2006,105021 10, 2006 Chemical Reviews, 111.11, 6557-6602, Aug. 2011. WO WO 2007/005874 1, 2007 Evans, et al., Asymmetric alkylation reactions of chiral imide WO WO 2008,1326O1 11/2008 enolates. A practical approach to the enantioselective synthesis of WO WO 2009/009 116 1, 2009 alpha-Substituted carboxylic acid derivatives, Journal of the Ameri WO WO 2009/044273 4/2009 can Chemical Society, 104(6), pp. 1737-1739, Mar. 1982. WO WO 2009/052320 4/2009 WO WO 2010.015655 2, 2010 Chiang et al. An Fc Domain Protein-Small Molecule Conjugate as WO WO 2010/O19570 2, 2010 an Enhanced Immunomodular, Journal of the American Chemical WO WO 2010/075376 T 2010 Society, 136(9):3370-3373, Feb. 2014. WO WO 2010/077634 T 2010 Li, W. et al., Current drug research on PEGylation with small WO WO 2011/028683 3, 2011 molecular agents, Progress in Polymer Science, 38:421-444, Apr. WO WO 2011/070024 6, 2011 2013. WO WO 2011/107553 9, 2011 Ramirez-Montagut et al., Immunity to melanoma: unraveling the WO WO 2011/109400 9, 2011 relation of tumor immunity and autoimmunity, Oncogene, WO WO 2011, 131407 10, 2011 WO WO 2011 140249 11, 2011 22(20):3180-3 187, May 2003. WO WO 2012/032433 3, 2012 Sawaya et al., Risk of cervical cancer associated with extending the WO WO 2012,087699 6, 2012 interval between cervical-cancer Screenings, New England Journal WO WO 2012/145493 10, 2012 of Medicine, 349(16): 1501-1509, Oct. 2003. WO WO 2013/07.9174 6, 2013 Pardoll. The blockade of immune checkpoints in cancer WO WO 2013,0794.25 6, 2013 immunotherapy, Nature Reviews Cancer, 12(4):252-264, Apr. 2012. WO WO 2013,119716 8, 2013 Sarkar, et al., Induction of indoleamine 2, 3-dioxygenase by inter WO WO 2013,132044 9, 2013 feron-y in human islets, Diabetes, 56(1):72-79, Jan. 2007. WO WO 2013, 169264 11 2013 Littlejohn, et al., Expression and Purification of Recombinant WO WO 2014.OO8218 1 2014 Human Indoleamine 2,3-Dioxygenase, Protein Expression and Puri WO WO 2014/036357 3, 2014 fication, 19(1):22-29, Jun. 2000. WO WO 2014/036412 3, 2014 Fox, et al., Discovery of 6-phenylpyrimido (4,5-b]14 oxazines as WO WO 2014/150677 9, 2014 potent and selective acyl CoA: diacylglycerol acyltransferase 1 WO WO 2014, 160967 10/2014 WO WO 2015, 188O85 12/2015 (DGAT1) inhibitors with in vivo efficacy in rodents, Journal of WO WO 2016/071283 5, 2016 Medical Chemistry, 57(8):3464-3483, Apr. 2014. WO WO 2016/073738 5, 2016 Yamamoto, et al., Additional reaction of arylboronic acid to alde WO WO 2016/07377O 5, 2016 hydes and O.f3-unsaturated carbonyl compounds catalyzed by con WO WO 2016/073774 5, 2016 ventional palladium complexes in the presence of chloroform, J Organomet, Chem. 69(9)4:1325-1332, Apr. 2009. Li, G. et al., Discovery of novel orally active ureido NPY Y5 OTHER PUBLICATIONS receptor antagonists, Bioorganic & Medical Chemistry Letters, 18(3): 1146-1150, Feb. 2008. 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FIG. A
Potency Potency C
NH O
CN
NH O
F
NH O
\ O
NH O U.S. Patent Mar. 21, 2017 Sheet 2 of 4 US 9,598.422 B2
FIG. B.
Potency Potency
H B H B N CN N C S O O)
CN H A C N N '-O-9 O O
N
O O
C H H N - A '-O- F A O O C C U.S. Patent Mar. 21, 2017 Sheet 3 of 4 US 9,598.422 B2
FIG. 1C
F Potency Potency C
A H NH C NO
C
O
C C
A H
O)-NH C-C- O
C
) A
Potency Potency U.S. Patent Mar. 21, 2017 Sheet 4 of 4 US 9,598.422 B2
FIG. D C Potency C. Potency
F C
Y-O-O.)FC '-O-O-)-.FC
CN CN
H
OC-)O C-C-CO US 9,598.422 B2 1. 2 MMUNOREGULATORY AGENTS not as well defined, IDO inhibitors may also find use in the treatment of patients with neurological or neuropsychiatric CROSS-REFERENCE TO RELATED diseases or disorders (e.g., depression). APPLICATIONS Small molecule inhibitors of IDO have been developed to treat or prevent IDO-related diseases. For example, the IDO This application claims priority to U.S. Provisional Appli inhibitors 1-methyl-DL-tryptophan; p-(3-benzofuranyl)- cation Ser. No. 62/075,678, filed Nov. 5, 2014, the entire DL-alanine; p-3-benzo(b)thienyl-DL-alanine; and 6-nitro content of which is incorporated herein by reference. L-tryptophan have been used to modulate T cell-mediated immunity by altering local extracellular concentrations of BACKGROUND OF THE INVENTION 10 tryptophan and tryptophan metabolites (WO 99/29310). Compounds having IDO inhibitory activity are further Indoleamine 2,3-dioxygenase (IDO; also known as IDO1) reported in PCT Publication No. WO 2004/094409. is an IFN-Y target gene that plays a role in immunomodu In view of the role played by indoleamine 2,3-dioxy lation. IDO is an oxidoreductase and one of two enzymes genase in a diverse array of diseases, disorders and condi that catalyze the first and rate-limiting step in the conversion 15 tions, and the limitations (e.g., efficacy) of current IDO of tryptophan to N-formyl-kynurenine. It exists as a 41 kD inhibitors, new IDO modulators, and compositions and monomer that is found in several cell populations, including methods associated therewith, are needed. immune cells, endothelial cells, and fibroblasts. IDO is relatively well-conserved between species, with mouse and BRIEF SUMMARY OF THE INVENTION human sharing 63% sequence identity at the amino acid level. Data derived from its crystal structure and site The present invention relates to compounds that modulate directed mutagenesis show that both Substrate binding and the oxidoreductase enzyme indoleamine 2,3-dioxygenase the relationship between the substrate and iron-bound dioxy (IDO), and compositions (e.g., pharmaceutical composi genase are necessary for activity. A homolog to IDO (IDO2) tions) comprising the compounds. Such compounds, includ has been identified that shares 44% amino acid sequence 25 ing methods of their synthesis, and compositions are homology with IDO, but its function is largely distinct from described in detail below. that of IDO. (See, e.g., Serafini, P. et al., Semin. Cancer The present invention also relates to the use of such Biol., 16(1):53-65 (February 2006) and Ball, H. J. et al., compounds and compositions for the treatment and/or pre Gene, 396(1):203-213 (Jul. 1, 2007)). vention of a diverse array of diseases, disorders and condi IDO plays a major role in immune regulation, and its 30 tions mediated, in whole or in part, by IDO. Such diseases, immunosuppressive function manifests in several manners. disorders and conditions are described in detail elsewhere Importantly, IDO regulates immunity at the T cell level, and herein. Unless otherwise indicated, when uses of the com a nexus exists between IDO and cytokine production. In pounds of the present invention are described herein, it is to addition, tumors frequently manipulate immune function by be understood that such compounds may be in the form of upregulation of IDO. Thus, modulation of IDO can have a 35 a composition (e.g., a pharmaceutical composition). therapeutic impact on a number of diseases, disorders and As discussed hereafter, although the compounds of the conditions. present invention are believed to effect their activity by A pathophysiological link exists between IDO and cancer. inhibition of IDO, a precise understanding of the com Disruption of immune homeostasis is intimately involved pounds underlying mechanism of action is not required to with tumor growth and progression, and the production of 40 practice the invention. It is envisaged that the compounds IDO in the tumor microenvironment appears to aid in tumor may alternatively effect their activity through inhibition of growth and metastasis. Moreover, increased levels of IDO tryptophan-2,3-dioxygenase (TDO) activity. It is also envis activity are associated with a variety of different tumors aged that the compounds may effect their activity through (Brandacher, G. et al., Clin. Cancer Res., 12(4): 1144-1151 inhibition of both IDO and TDO function. Although the (Feb. 15, 2006)). 45 compounds of the invention are generally referred to herein Treatment of cancer commonly entails Surgical resection as IDO inhibitors, it is to be understood that the term “IDO followed by chemotherapy and radiotherapy. The standard inhibitors' encompasses compounds that act individually treatment regimens show highly variable degrees of long through inhibition of TDO or IDO, and/or compounds that term success because of the ability of tumor cells to essen act through inhibition of both IDO and TDO. tially escape by regenerating primary tumor growth and, 50 In one aspect, the present invention provides compounds often more importantly, seeding distant metastasis. Recent represented by formula (I): advances in the treatment of cancer and cancer-related diseases, disorders and conditions comprise the use of (I) combination therapy incorporating immunotherapy with more traditional chemotherapy and radiotherapy. Under 55 most scenarios, immunotherapy is associated with less tox icity than traditional chemotherapy because it utilizes the patient’s own immune system to identify and eliminate tumor cells. In addition to cancer, IDO has been implicated in, among 60 other conditions, immunosuppression, chronic infections, and autoimmune diseases or disorders (e.g., rheumatoid or a pharmaceutically acceptable salt, hydrate or Solvate arthritis). Thus, Suppression of tryptophan degradation by thereof, wherein, inhibition of IDO activity has tremendous therapeutic value. the subscript n is 1 or 0; Moreover, inhibitors of IDO can be used to enhance T cell 65 A is C(O)—, NH SO , CH2—, or activation when the T cells are Suppressed by pregnancy, CHR ; malignancy, or a virus (e.g., HIV). Although their roles are B is a bond, —C(O) , NH , —CH2—, or —CHR : US 9,598.422 B2 3 4 T is a bond, CH —NH , O s OCH , the cancer is melanoma, colon cancer, pancreatic cancer, —C(O)CH , or –CRR : breast cancer, prostate cancer, lung cancer, leukemia, a brain wherein when A is NH and B is —C(O)—, then T is tumor, lymphoma, Sarcoma, ovarian cancer, head and neck other than –C(R)(R)-; D is N or C(R): cancer, cervical cancer, or Kaposi's sarcoma. Cancers that E is N or C(R): are candidates for treatment with the compounds and com V is a bond, O , or C(R): positions of the present invention are discussed further G is an optionally substituted aryl, optionally substituted hereafter. heteroaryl, or an optionally substituted 9- or 10-mem The present invention contemplates methods of treating a bered fused bicyclic heteroaryl; Subject receiving a bone marrow transplant or peripheral J" is CH, N or C(R), when R is attached to the ring vertex 10 identified as J'; blood stem cell transplant by administering a therapeutically R" and Rare independently hydrogen, halogen, optionally effective amount of an IDO inhibitor sufficient to increase Substituted C-C haloalkyl, optionally substituted C-C, the delayed-type hypersensitivity reaction to tumor antigen, cycloalkyl, optionally substituted 3- to 6-membered delay the time-to-relapse of post-transplant malignancy, cycloheteroalkyl, optionally substituted phenyl, option 15 increase relapse-free Survival time post-transplant, and/or ally substituted heteroaryl, optionally substituted C-C, increase long-term post-transplant Survival. alkyl, optionally substituted C-C alkoxy, CN, SONH2, In certain embodiments, the present invention contem NHSOCH, NHSOCF. OCF, SOCH, SOCF, or plates methods for treating or preventing an infective dis CONH2, and when R' and Rare on adjacent vertices of order (e.g., a viral infection) in a subject (e.g., a human) a phenyl ring they may be joined together to form a 5- or comprising administering to the Subject a therapeutically 6-membered cycloheteroalkyl ring having one or two ring effective amount of at least one IDO inhibitor (e.g., a novel vertices independently selected from O, N and S, wherein inhibitor of the instant invention). In some embodiments, the said cycloheteroalkyl ring is optionally substituted with infective disorder is a viral infection (e.g., a chronic viral from one to three members selected from fluoro and infection), a bacterial infection, or a parasitic infection. In C-C alkyl, 25 certain embodiments, the viral infection is human immuno R and R are independently hydrogen, optionally substi deficiency virus or cytomegalovirus. In other embodiments, tuted C-C alkyl, optionally substituted C-C haloalkyl, the bacterial infection is a Mycobacterium infection (e.g., fluorine, OH, CN, COH, C(O)NH, N(R), optionally Mycobacterium leprae or Mycobacterium tuberculosis). In substituted —O-C-C alkyl, —(CRR), OH, still other embodiments, the parasitic infection is Leishma —(CRR), COH, -(CRR), C(O)NH, 30 (CRR), C(O)NHR', (CRR), N(R), NH nia donovani, Leishmania tropica, Leishmania major Leish (CRR), COH or NH(CRR), C(O)NH: mania aethiopica, Leishmania mexicana, Plasmodium fal each R is independently H, F, OH, optionally substituted ciparum, Plasmodium vivax, Plasmodium ovale, or C-C alkyl or optionally Substituted —O—C-C alkyl; Plasmodium malariae. In further embodiments, the infective each R is independently H, or optionally substituted C-C, 35 disorder is a fungal infection. alkyl: In still other embodiments, the present invention contem R is H, OH, F, optionally substituted C-C alkyl, optionally plates methods for treating or preventing an immune-related substituted —O C-C alkyl, or N(R): disease, disorder or condition in a Subject (e.g., a human), and each m is independently 1, 2, or 3. comprising administering to the Subject a therapeutically In yet another aspect, the present invention provides 40 effective amount of at least one IDO inhibitor (e.g., prefer compositions in which compounds of formula (I), are com ably a novel inhibitor of the instant invention). Examples of bined with one or more pharmaceutically acceptable excipi immune-related diseases, disorders and conditions are entS. described hereafter. In some embodiments, the present invention contemplates Other diseases, disorders and conditions that may be methods for treating or preventing cancer in a Subject (e.g., 45 treated or prevented, in whole or in part, by modulation of a human) comprising administering to the Subject a thera IDO activity are candidate indications for the IDO inhibitor peutically effective amount of at least one IDO inhibitor compounds that are described herein. described herein. The present invention includes methods of The present invention further contemplates the use of the treating or preventing a cancer in a Subject by administering IDO inhibitors described herein in combination with one or to the subject an IDO inhibitor in an amount effective to 50 more additional agents. The one or more additional agents reverse or stop the progression of IDO-mediated immuno may have some IDO modulating activity and/or they may Suppression. In some embodiments, the IDO-mediated function through distinct mechanisms of action. In some immunosuppression is mediated by an antigen-presenting embodiments, such agents comprise radiation (e.g., local cell (APC). ized radiation therapy or total body radiation therapy) and/or Examples of the cancers that may be treated using the 55 other treatment modalities of a non-pharmacological nature. compounds and compositions described herein include, but When combination therapy is utilized, the IDO inhibitor(s) are not limited to: cancers of the prostate, colorectum, and the one additional agent(s) may be in the form of a single pancreas, cervix, stomach, endometrium, brain, liver, blad composition or multiple compositions, and the treatment der, ovary, testis, head, neck, skin (including melanoma and modalities may be administered concurrently, sequentially, basal carcinoma), mesothelial lining, white blood cell (in 60 or through some other regimen. By way of example, the cluding lymphoma and leukemia) esophagus, breast, present invention contemplates a treatment regimen wherein muscle, connective tissue, lung (including Small-cell lung a radiation phase is followed by a chemotherapeutic phase. carcinoma and non-Small-cell carcinoma), adrenal gland, The combination therapy may have an additive or synergis thyroid, kidney, or bone; glioblastoma, mesothelioma, renal tic effect. Other benefits of combination therapy are cell carcinoma, gastric carcinoma, sarcoma, choriocarci 65 described hereafter. noma, cutaneous basocellular carcinoma, and testicular In some embodiments, the present invention further com seminoma. In some embodiments of the present invention, prises the use of the IDO inhibitors described herein in US 9,598.422 B2 5 6 combination with bone marrow transplantation, peripheral tering either alone. In further embodiments drawn to meth blood stem cell transplantation, or other types of transplan ods of treating cancer, the administration of a therapeutically tation therapy. effective amount of an IDO inhibitor in combination with at In particular embodiments, the present invention contem least one chemotherapeutic agent results in a reduction of plates the use of the inhibitors of IDO function described tumor size or a slowing of tumor growth greater than herein in combination with immune checkpoint inhibitors. reduction of the tumor size or tumor growth observed by The blockade of immune checkpoints, which results in the administration of one agent alone. amplification of antigen-specific T cell responses, has been In further embodiments, the present invention contem shown to be a promising approach in human cancer thera plates methods for treating or preventing cancer in a Subject, peutics. Examples of immune checkpoints (ligands and 10 comprising administering to the Subject a therapeutically receptors). Some of which are selectively upregulated in effective amount of at least one IDO inhibitor and at least various types of tumor cells, that are candidates for blockade one signal transduction inhibitor (STI). In a particular include PD1 (programmed cell death protein 1); PDL1 (PD1 embodiment, the at least one STI is selected from the group ligand); BTLA (B and T lymphocyte attenuator); CTLA4 consisting of bcr/abl kinase inhibitors, epidermal growth (cytotoxic T-lymphocyte associated antigen 4); TIM3 (T-cell 15 factor (EGF) receptor inhibitors, her-2/neu receptor inhibi membrane protein 3); LAG3 (lymphocyte activation gene tors, and farnesyl transferase inhibitors (FTIs). Other can 3); A2aR (adenosine A2a receptor A2aR); and Killer Inhibi didate STI agents are set forth elsewhere herein. tory Receptors. Immune checkpoint inhibitors, and combi The present invention also contemplates methods of aug nation therapy therewith, are discussed in detail elsewhere menting the rejection of tumor cells in a Subject comprising herein. administering an IDO inhibitor in conjunction with at least In other embodiments, the present invention provides one chemotherapeutic agent and/or radiation therapy, methods for treating cancer in a Subject, comprising admin wherein the resulting rejection of tumor cells is greater than istering to the subject a therapeutically effective amount of that obtained by administering either the IDO inhibitor, the at least one IDO inhibitor and at least one chemotherapeutic chemotherapeutic agent or the radiation therapy alone. agent, such agents including, but not limited to alkylating 25 In further embodiments, the present invention provides agents (e.g., nitrogen mustards Such as chlorambucil, cyclo methods for treating cancer in a Subject, comprising admin phosphamide, isofamide, mechlorethamine, melphalan, and istering to the subject a therapeutically effective amount of uracil mustard; aziridines such as thiotepa, methaneSulpho at least one IDO inhibitor and at least one immunomodulator nate esters such as buSulfan, nucleoside analogs (e.g., gem other than an IDO inhibitor. In particular embodiments, the citabine); nitroso ureas such as carmustine, lomustine, and 30 at least one immunomodulator is selected from the group streptozocin, topoisomerase 1 inhibitors (e.g., irinotecan); consisting of CD40L, B7, B7RP1, ant-CD40, anti-CD38, platinum complexes such as cisplatin and carboplatin: biore anti-ICOS, 4-IBB ligand, dendritic cell cancer vaccine, IL2, ductive alkylators such as mitomycin, procarbazine, dacar IL12, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18, TNF, bazine and altretamine); DNA strand-breakage agents (e.g., IL-15, MDC, IFN-o/-3, M-CSF, IL-3, GM-CSF, IL-13, and bleomycin); topoisomerase II inhibitors (e.g., amsacrine, 35 anti-IL-10. Other candidate immunomodulator agents are set dactinomycin, daunorubicin, idarubicin, mitoxantrone, forth elsewhere herein. doxorubicin, etoposide, and teniposide); DNA minor groove The present invention contemplates embodiments com binding agents (e.g., plicamydin); antimetabolites (e.g., prising methods for treating or preventing an infective folate antagonists such as methotrexate and trimetrexate; disorder (e.g., a viral infection) in a subject (e.g., a human) pyrimidine antagonists such as fluorouracil, fluorodeoxyu 40 comprising administering to the Subject a therapeutically ridine, CB3717, azacitidine, cytarabine, and floxuridine: effective amount of at least one IDO inhibitor and a thera purine antagonists such as mercaptopurine, 6-thioguanine, peutically effective amount of an anti-infective agent(s) fludarabine, pentostatin; asparginase; and ribonucleotide In some embodiments of the present invention, the addi reductase inhibitors such as hydroxyurea); tubulin interac tional therapeutic agent is a cytokine, including, for tive agents (e.g., Vincristine, estramustine, vinblastine, doc 45 example, granulocyte-macrophage colony stimulating factor etaXol, epothilone derivatives, and paclitaxel); hormonal (GM-CSF) or flt3-ligand. The present invention also con agents (e.g., estrogens, conjugated estrogens, ethinyl estra templates methods for treating or preventing a viral infection diol; diethylstilbesterol: chlortrianisen; idenestrol; proges (e.g., a chronic viral infection) including, but not limited to, tins such as hydroxyprogesterone caproate, medroxyproges hepatitis C virus (HCV), human papilloma virus (HPV), terone, and megestrol; and androgens such as testosterone, 50 cytomegalovirus (CMV), Epstein-Barr virus (EBV), vari testosterone propionate, fluoxymesterone, and methyltestos cella Zoster virus, coxsackie virus, and human immunode terone); adrenal corticosteroids (e.g., prednisone, dexam ficiency virus (HIV). The use of the IDO inhibitors ethasone, methylprednisolone, and prednisolone); leutiniz described herein to treat (either alone or as a component of ing hormone releasing agents or gonadotropin-releasing combination therapy) infection is discussed further hereaf hormone antagonists (e.g., leuprolide acetate and goserelin 55 ter. acetate); and antihormonal antigens (e.g., tamoxifen, anti In additional embodiments, treatment of an infective androgen agents such as flutamide; and antiadrenal agents disorder is effected through the co-administration of a vac Such as mitotane and aminoglutethimide). The present cine in combination with administration of a therapeutically invention also contemplates the use of the IDO inhibitors in effective amount of an IDO inhibitor of the present inven combination with other agents known in the art (e.g., arsenic 60 tion. In some embodiments, the vaccine is an anti-viral trioxide) and other chemotherapeutic agents developed in vaccine, including, for example, an anti-HIV vaccine. In the future. other embodiments, the vaccine is effective against tuber In some embodiments drawn to methods of treating culosis or malaria. In still other embodiments, the vaccine is cancer, the administration of a therapeutically effective a tumor vaccine (e.g., a vaccine effective against mela amount of an IDO inhibitor in combination with at least one 65 noma); the tumor vaccine may comprise genetically modi chemotherapeutic agent results in a cancer Survival rate fied tumor cells or a genetically modified cell line, including greater than the cancer Survival rate observed by adminis genetically modified tumor cells or a genetically modified US 9,598.422 B2 7 8 cell line that has been transfected to express granulocyte comprising chemotherapy and radiotherapy are generally macrophage stimulating factor (GM-CSF). In particular difficult for the patient to tolerate and become less effective embodiments, the vaccine includes one or more immuno as tumors evolve to survive such treatments. By utilizing the genic peptides and/or dendritic cells. patient's own immune system to identify and eliminate In some embodiments, the present invention contemplates tumor cells, immunotherapy has the benefit of reduced methods of using the IDO inhibitors disclosed herein in toxicity. As upregulation of the immunoregulatory enzyme combination with one or more antimicrobial agents. indoleamine 2,3-dioxygenase comprises one mechanism In certain embodiments drawn to treatment of an infection manipulated by tumors to promote growth, agents (e.g., by administering an IDO inhibitor and at least one additional Small molecule compounds) that inhibit enzyme activity therapeutic agent, a symptom of infection observed after 10 present a promising avenue for prophylaxis and/or treat administering both the IDO inhibitor and the additional ment. therapeutic agent is improved over the same symptom of In addition, a large body of experimental data indicates a infection observed after administering either alone. In some role for IDO inhibition in immunosuppression, tumor resis embodiments, the symptom of infection observed may be tance and/or rejection, chronic infections, HIV-infection, reduction in viral load, increase in CD4 T cell count, 15 and autoimmune diseases or disorders. Inhibition of IDO decrease in opportunistic infections, increased Survival time, may also be an important treatment strategy for patients with eradication of chronic infection, or a combination thereof. neurological or neuropsychiatric diseases or disorders such as depression. The compounds, compositions and methods BRIEF DESCRIPTION OF THE DRAWINGS herein address the need for new classes of IDO modulators. FIGS. 1A, 1B, 1C and 1D provide structures and biologi Definitions cal activity for compounds described herein. Unless otherwise indicated, the following terms are DETAILED DESCRIPTION OF THE intended to have the meaning set forth below. Other terms INVENTION 25 are defined elsewhere throughout the specification. The term “alkyl, by itself or as part of another substitu Before the present invention is further described, it is to ent, means, unless otherwise stated, a straight or branched be understood that the invention is not limited to the chain hydrocarbon radical, having the number of carbon particular embodiments set forth herein, and it is also to be atoms designated (i.e., C.s means one to eight carbons). understood that the terminology used herein is for the 30 Examples of alkyl groups include methyl, ethyl, n-propyl. purpose of describing particular embodiments only, and is isopropyl. n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, not intended to be limiting. n-hexyl, n-heptyl, n-octyl, and the like. Where a range of values is provided, it is understood that The term “cycloalkyl refers to hydrocarbon rings having each intervening value, to the tenth of the unit of the lower the indicated number of ring atoms (e.g., C. cycloalkyl) limit unless the context clearly dictates otherwise, between 35 and being fully saturated or having no more than one double the upper and lower limit of that range and any other stated bond between ring vertices. “Cycloalkyl is also meant to or intervening value in that stated range, is encompassed refer to bicyclic and polycyclic hydrocarbon rings such as, within the invention. The upper and lower limits of these for example, bicyclo[2.2.1]heptane, bicyclo2.2.2]octane, Smaller ranges may independently be included in the Smaller etc. ranges, and are also encompassed within the invention, 40 The term “cycloheteroalkyl refers to a cycloalkyl ring Subject to any specifically excluded limit in the Stated range. having the indicated number of ring vertices (or members) Where the stated range includes one or both of the limits, and having from one to five heteroatoms selected from N, O, ranges excluding either or both of those included limits are and S, which replace one to five of the carbon vertices, and also included in the invention. Unless defined otherwise, all wherein the nitrogen and Sulfur atoms are optionally oxi technical and Scientific terms used herein have the same 45 dized, and the nitrogen atom(s) are optionally quaternized. meaning as commonly understood by one of ordinary skill The cycloheteroalkyl may be a monocyclic, a bicyclic or a in the art to which this invention belongs. polycyclic ring system. Non limiting examples of cyclohet It must be noted that as used herein and in the appended eroalkyl groups include pyrrolidine, imidazolidine, pyrazo claims, the singular forms “a”, “an', and “the include plural lidine, butyrolactam, Valerolactam, imidazolidinone, hydan referents unless the context clearly dictates otherwise. It is 50 toin, dioxolane, phthalimide, piperidine, 1,4-dioxane, further noted that the claims may be drafted to exclude any morpholine, thiomorpholine, thiomorpholine-S-oxide, thio optional element. As such, this statement is intended to serve morpholine-SS-oxide, piperazine, pyran, pyridone, 3-pyr as antecedent basis for use of Such exclusive terminology roline, thiopyran, pyrone, tetrahydrofuran, tetrhydrothio such as “solely”, “only' and the like in connection with the phene, quinuclidine, and the like. A cycloheteroalkyl group recitation of claim elements, or use of a “negative' limita 55 can be attached to the remainder of the molecule through a tion. ring carbon or a heteroatom. The publications discussed herein are provided solely for As used herein, a wavy line, “W, that intersects a their disclosure prior to the filing date of the present appli single, double or triple bond in any chemical structure cation. Further, the dates of publication provided may be depicted herein, represent the point attachment of the single, different from the actual publication dates, which may need 60 double, or triple bond to the remainder of the molecule. to be independently confirmed. Additionally, a bond extending to the center of a ring (e.g., a phenyl ring) is meant to indicate attachment at any of the General available ring vertices. One of skill in the art will understand that multiple Substituents shown as being attached to a ring Immune dysregulation is intimately associated with tumor 65 will occupy ring vertices that provide stable compounds and evasion of the host immune system, resulting in tumor are otherwise sterically compatible. For a divalent compo growth and progression. Traditional treatment approaches nent, a representation is meant to include either orientation US 9,598.422 B2 9 10 (forward or reverse). For example, the group " C(O) nitrogen atom, they can be combined with the nitrogenatom NH is meant to include a linkage in either orientation: to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, —C(O)NH or —NHC(O)—, and similarly, “ O— —NR'R'" is meant to include 1-pyrrolidinyl and 4-morpholi CHCH is meant to include both —O—CH2CH2—and nyl. —CH2CH2—O—. Similarly, optional substituents for the aryland heteroaryl The terms “alkoxy”, “alkylamino” and “alkylthio’ (or groups are varied and are generally selected from: -halogen, thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a Sulfur atom, respectively. Additionally, for dialkylamino groups, the 10 NH CONH)—NH, NR'C(NH)—NH, -NH C alkyl portions can be the same or different and can also be (NH)—NR', S(O)R', S(O).R', S(O)NR'R", NR'S combined to form a 3-7 membered ring with the nitrogen (O)R", —N, perfluoro(C-C)alkoxy, and perfluoro(C- atom to which each is attached. Accordingly, a group rep C.)alkyl, in a number ranging from Zero to the total number resented as dialkylamino or - NR'R' is meant to include of open Valences on the aromatic ring system; and where R', piperidinyl, pyrrolidinyl, morpholinyl, aZetidinyl and the 15 R" and R" are independently selected from hydrogen, Cs like. alkyl, Cs haloalkyl, C. cycloalkyl, Cs alkenyl and Cs The terms “halo' or “halogen, by themselves or as part alkynyl. Other suitable substituents include each of the of another Substituent, mean, unless otherwise stated, a above aryl Substituents attached to a ring atom by an fluorine, chlorine, bromine, or iodine atom. Additionally, alkylene tether of from 1-4 carbon atoms. terms such as "haloalkyl, are meant to include monoha Two of the substituents on adjacent atoms of the aryl or loalkyl and polyhaloalkyl. For example, the term "Ca heteroaryl ring may optionally be replaced with a substituent haloalkyl is mean to include trifluoromethyl, 2.2.2-trifluo of the formula -T-C(O) (CH), U , wherein T and U roethyl, 4-chlorobutyl, 3-bromopropyl, and the like. are independently —NH-, -O-, -CH2—, or a single The term “aryl' means, unless otherwise stated, a poly bond, and q is an integer of from 0 to 2. Alternatively, two unsaturated, typically aromatic, hydrocarbon group which 25 of the substituents on adjacent atoms of the aryl or heteroaryl can be a single ring or multiple rings (up to three rings) ring may optionally be replaced with a Substituent of the which are fused together or linked covalently. Non-limiting formula -A-(CH2), B–, wherein A and B are indepen examples of aryl groups include phenyl, naphthyl and biphe dently —CH2—, —O— —NH-. —S , —S(O)—, nyl. —S(O) , —S(O)NR' - or a single bond, and r is an The term "heteroaryl” refers to aryl groups (or rings) that 30 integer of from 1 to 3. One of the single bonds of the new contain from one to five heteroatoms selected from N, O, ring so formed may optionally be replaced with a double and S, wherein the nitrogen and sulfur atoms are optionally bond. Alternatively, two of the substituents on adjacent oxidized, and the nitrogen atom(s) are optionally quater atoms of the aryl or heteroaryl ring may optionally be nized. A heteroaryl group can be attached to the remainder replaced with a substituent of the formula —(CH), X of the molecule through a heteroatom. Non-limiting 35 (CH) , where S and t are independently integers of from examples of heteroaryl groups include pyridyl, pyridazinyl, 0 to 3, and X is —O— —NR' , —S —S(O)—, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, —S(O) , or —S(O)NR' . The substituent R' in quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, puri —NR'— and —S(O)NR' is selected from hydrogen or nyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, ben unsubstituted C. alkyl. Zisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotri 40 As used herein, the term "heteroatom' is meant to include azinyl, thienopyridinyl, thienopyrimidinyl, oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). pyrazolopyrimidinyl, imidazopyridines, benzothiaxolyl, The term “pharmaceutically acceptable salts' is meant to benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, include salts of the active compounds which are prepared isothiazolyl pyrazolyl, indazolyl, pteridinyl, imidazolyl, tri with relatively nontoxic acids or bases, depending on the azolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl pyrro 45 particular Substituents found on the compounds described lyl, thiazolyl, furyl, thienyl and the like. Substituents for a herein. When compounds of the present invention contain heteroaryl ring can be selected from the group of acceptable relatively acidic functionalities, base addition salts can be substituents described below. obtained by contacting the neutral form of Such compounds The above terms (e.g., “alkyl”, “aryl and "heteroaryl'), with a sufficient amount of the desired base, either neat or in in some embodiments, will be optionally substituted. 50 a suitable inert solvent. Examples of salts derived from Selected substituents for each type of radical are provided pharmaceutically acceptable inorganic bases include alumi below. num, ammonium, calcium, copper, ferric, ferrous, lithium, Optional substituents for the alkyl radicals (including magnesium, manganic, manganous, potassium, Sodium, Zinc those groups often referred to as alkylene, alkenyl, alkynyl and the like. Salts derived from pharmaceutically acceptable and cycloalkyl) can be a variety of groups selected from: 55 organic bases include salts of primary, secondary and ter halogen, OR', NR'R'', SR', SiR"R"R", OC(O)R', tiary amines, including Substituted amines, cyclic amines, C(O)R, COR, CONR'R", OC(O)NR'R", naturally-occurring amines and the like, Such as arginine, NR"C(O)R', NR C(O)NR"R", NR"C(O).R', betaine, caffeine, choline, N,N'-dibenzylethylenediamine, NH CONH)—NH, NR'C(NH)—NH, -NH C diethylamine, 2-diethylaminoethanol, 2-dimethylamin (NH)—NR', S(O)R', S(O).R', S(O)NR'R", NR'S 60 oethanol, ethanolamine, ethylenediamine, N-ethylmorpho (O).R", —CN and - NO, in a number ranging from Zero to line, N-ethylpiperidine, glucamine, glucosamine, histidine, (2m'+1), where m' is the total number of carbon atoms in hydrabamine, isopropylamine, lysine, methylglucamine, such radical. R', R" and R" each independently refer to morpholine, piperazine, piperidine, polyamine resins, pro hydrogen, unsubstituted Cls alkyl, unsubstituted aryl, aryl caine, purines, theobromine, triethylamine, trimethylamine, Substituted with 1-3 halogens, unsubstituted Cls alkyl, Cs 65 tripropylamine, tromethamine and the like. When com alkoxy or Cs thioalkoxy groups, or unsubstituted aryl-Ca pounds of the present invention contain relatively basic alkyl groups. When R' and R" are attached to the same functionalities, acid addition salts can be obtained by con US 9,598.422 B2 11 12 tacting the neutral form of Such compounds with a Sufficient tium (H), iodine-125 ('I) or carbon-14 (''C), or non amount of the desired acid, either neat or in a suitable inert radioactive isotopes, such as deuterium (H) or carbon-13 Solvent. Examples of pharmaceutically acceptable acid addi (C). Such isotopic variations can provide additional utili tion salts include those derived from inorganic acids like ties to those described elsewhere within this application. For hydrochloric, hydrobromic, nitric, carbonic, monohydro instance, isotopic variants of the compounds of the invention gencarbonic, phosphoric, monohydrogenphosphoric, dihy may find additional utility, including but not limited to, as drogenphosphoric, Sulfuric, monohydrogensulfuric, hydri diagnostic and/or imaging reagents, or as cytotoxic/radio odic, or phosphorous acids and the like, as well as the salts toxic therapeutic agents. Additionally, isotopic variants of derived from relatively nontoxic organic acids like acetic, the compounds of the invention can have altered pharma propionic, isobutyric, malonic, benzoic. Succinic, Suberic, 10 cokinetic and pharmacodynamic characteristics which can fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfo contribute to enhanced safety, tolerability or efficacy during nic, citric, tartaric, methanesulfonic, and the like. Also treatment. All isotopic variations of the compounds of the included are salts of amino acids such as arginate and the present invention, whether radioactive or not, are intended to like, and salts of organic acids like glucuronic or galac be encompassed within the scope of the present invention. tunoric acids and the like (see, for example, Berge, S. M. et 15 The terms “patient’ or “subject' are used interchangeably al., “Pharmaceutical Salts”, J. Pharm. Sci., 66:1-19 (1977)). to refer to a human or a non-human animal (e.g., a mammal). Certain specific compounds of the present invention contain The terms 'administration', 'administer” and the like, as both basic and acidic functionalities that allow the com they apply to, for example, a Subject, cell, tissue, organ, or pounds to be converted into either base or acid addition salts. biological fluid, refer to contact of for example, an inhibitor The neutral forms of the compounds may be regenerated of IDO, a pharmaceutical composition comprising same, or by contacting the salt with a base or acid and isolating the a diagnostic agent to the Subject, cell, tissue, organ, or parent compound in the conventional manner. The parent biological fluid. In the context of a cell, administration form of the compound differs from the various salt forms in includes contact (e.g., in vitro or ex vivo) of a reagent to the certain physical properties, such as solubility in polar Sol cell, as well as contact of a reagent to a fluid, where the fluid vents, but otherwise the salts are equivalent to the parent 25 is in contact with the cell. form of the compound for the purposes of the present The terms “treat”, “treating”, “treatment' and the like invention. refer to a course of action (Such as administering an inhibitor In addition to Salt forms, the present invention provides of IDO or a pharmaceutical composition comprising same) compounds which are in a prodrug form. Prodrugs of the initiated after a disease, disorder or condition, or a symptom compounds described herein are those compounds that read 30 thereof, has been diagnosed, observed, and the like so as to ily undergo chemical changes under physiological condi eliminate, reduce, Suppress, mitigate, or ameliorate, either tions to provide the compounds of the present invention. temporarily or permanently, at least one of the underlying Additionally, prodrugs can be converted to the compounds causes of a disease, disorder, or condition afflicting a subject, of the present invention by chemical or biochemical meth or at least one of the symptoms associated with a disease, ods in an ex vivo environment. For example, prodrugs can 35 disorder, condition afflicting a Subject. Thus, treatment be slowly converted to the compounds of the present inven includes inhibiting (e.g., arresting the development or fur tion when placed in a transdermal patch reservoir with a ther development of the disease, disorder or condition or Suitable enzyme or chemical reagent. clinical symptoms association therewith) an active disease. Certain compounds of the present invention can exist in The term “in need of treatment” as used herein refers to unsolvated forms as well as Solvated forms, including 40 a judgment made by a physician or other caregiver that a hydrated forms. In general, the Solvated forms are equivalent subject requires or will benefit from treatment. This judg to unsolvated forms and are intended to be encompassed ment is made based on a variety of factors that are in the within the scope of the present invention. Certain com realm of the physician’s or caregiver's expertise. pounds of the present invention may exist in multiple The terms “prevent”, “preventing”, “prevention' and the crystalline or amorphous forms. In general, all physical 45 like refer to a course of action (Such as administering an IDO forms are equivalent for the uses contemplated by the inhibitor or a pharmaceutical composition comprising same) present invention and are intended to be within the scope of initiated in a manner (e.g., prior to the onset of a disease, the present invention. disorder, condition or symptom thereof) So as to prevent, Certain compounds of the present invention possess Suppress, inhibit or reduce, either temporarily or perma asymmetric carbon atoms (optical centers) or double bonds; 50 nently, a Subject's risk of developing a disease, disorder, the racemates, diastereomers, geometric isomers, regioiso condition or the like (as determined by, for example, the mers and individual isomers (e.g., separate enantiomers) are absence of clinical symptoms) or delaying the onset thereof, all intended to be encompassed within the scope of the generally in the context of a Subject predisposed to having present invention. When a stereochemical depiction is a particular disease, disorder or condition. In certain shown, it is meant to refer the compound in which one of the 55 instances, the terms also refer to slowing the progression of isomers is present and substantially free of the other isomer. the disease, disorder or condition or inhibiting progression “Substantially free of another isomer indicates at least an thereof to a harmful or otherwise undesired state. 80/20 ratio of the two isomers, more preferably 90/10, or The term “in need of prevention” as used herein refers to 95/5 or more. In some embodiments, one of the isomers will a judgment made by a physician or other caregiver that a be present in an amount of at least 99%. 60 subject requires or will benefit from preventative care. This The compounds of the present invention may also contain judgment is made based on a variety of factors that are in the unnatural proportions of atomic isotopes at one or more of realm of a physicians or caregiver's expertise. the atoms that constitute such compounds. Unnatural pro The phrase “therapeutically effective amount” refers to portions of an isotope may be defined as ranging from the the administration of an agent to a subject, either alone or as amount found in nature to an amount consisting of 100% of 65 part of a pharmaceutical composition and either in a single the atom in question. For example, the compounds may dose or as part of a series of doses, in an amount capable of incorporate radioactive isotopes, such as, for example, tri having any detectable, positive effect on any symptom, US 9,598.422 B2 13 14 aspect, or characteristic of a disease, disorder or condition increase, activate, facilitate, enhance activation, sensitize, or when administered to the subject. The therapeutically effec up-regulate, e.g., a gene, protein, ligand, receptor, or cell. An tive amount can be ascertained by measuring relevant physi inhibitor may also be defined as a molecule that reduces, ological effects, and it can be adjusted in connection with the blocks, or inactivates a constitutive activity. An "agonist' is dosing regimen and diagnostic analysis of the Subjects a molecule that interacts with a target to cause or promote an condition, and the like. By way of example, measurement of increase in the activation of the target. An 'antagonist' is a the serum level of and IDO inhibitor (or, e.g., a metabolite molecule that opposes the action(s) of an agonist. An antago thereof) at a particular time post-administration may be nist prevents, reduces, inhibits, or neutralizes the activity of indicative of whether a therapeutically effective amount has an agonist, and an antagonist can also prevent, inhibit, or been used. 10 reduce constitutive activity of a target, e.g., a target receptor, The phrase “in a sufficient amount to effect a change' even where there is no identified agonist. means that there is a detectable difference between a level of The terms “modulate”, “modulation' and the like refer to an indicator measured before (e.g., a baseline level) and after the ability of a molecule (e.g., an activator or an inhibitor) administration of a particular therapy. Indicators include any to increase or decrease the function or activity of IDO, either objective parameter (e.g., serum concentration) or Subjective 15 directly or indirectly. A modulator may act alone, or it may parameter (e.g., a subjects feeling of well-being). use a cofactor, e.g., a protein, metal ion, or Small molecule. The term “small molecules' refers to chemical com Examples of modulators include Small molecule compounds pounds having a molecular weight that is less than about 10 and other bioorganic molecules. Numerous libraries of small kDa, less than about 2 kDa, or less than about 1 kDa. Small molecule compounds (e.g., combinatorial libraries) are com molecules include, but are not limited to, inorganic mol mercially available and can serve as a starting point for ecules, organic molecules, organic molecules containing an identifying a modulator. The skilled artisan is able to inorganic component, molecules comprising a radioactive develop one or more assays (e.g., biochemical or cell-based atom, and synthetic molecules. Therapeutically, a small assays) in which Such compound libraries can be screened in molecule may be more permeable to cells, less Susceptible order to identify one or more compounds having the desired to degradation, and less likely to elicit an immune response 25 properties; thereafter, the skilled medicinal chemist is able to than large molecules. optimize such one or more compounds by, for example, As used herein, the terms “IDO inhibitor”, “IDO blocker' synthesizing and evaluating analogs and derivatives thereof. and terms similar thereto refer to agents capable of inhibit Synthetic and/or molecular modeling studies can also be ing the activity of IDO, thereby reversing IDO-mediated utilized in the identification of an Activator. immunosuppression. An IDO inhibitor may be a competi 30 The “activity” of a molecule may describe or refer to the tive, noncompetitive, or irreversible IDO inhibitor. “A com binding of the molecule to a ligand or to a receptor, to petitive IDO inhibitor” is a compound that reversibly inhib catalytic activity; to the ability to stimulate gene expression its IDO enzyme activity at the catalytic site: “a or cell signaling, differentiation, or maturation; to antigenic noncompetitive IDO Inhibitor is a compound that revers activity; to the modulation of activities of other molecules: ibly inhibits IDO enzyme activity at a non-catalytic site; and 35 and the like. The term “proliferative activity” encompasses “an irreversible IDO inhibitor is a compound that irrevers an activity that promotes, that is necessary for, or that is ibly eliminates IDO enzyme activity by forming a covalent specifically associated with, for example, normal cell divi bond (or other stable means of inhibiting enzyme function) Sion, as well as cancer, tumors, dysplasia, cell transforma with the enzyme. A number of IDO inhibitors are commer tion, metastasis, and angiogenesis. cially available (e.g., 5-Br-4-C1-indoxyl 1,3-diacetate and 40 As used herein, “comparable”, “comparable activity”, 1-methyl-DL-tryptophan (1 MT); both available from “activity comparable to”, “comparable effect”, “effect com Sigma-Aldrich, St. Louis, Mo.) and may be used as, for parable to’, and the like are relative terms that can be viewed example, “tool” or “reference’ compounds quantitatively and/or qualitatively. The meaning of the terms The term “ligand’ refers to, for example, a peptide, a is frequently dependent on the context in which they are polypeptide, a membrane-associated or membrane-bound 45 used. By way of example, two agents that both activate a molecule, or a complex thereof, that can act as an agonist or receptor can be viewed as having a comparable effect from antagonist of a receptor. A ligand encompasses natural and a qualitative perspective, but the two agents can be viewed Synthetic ligands, e.g., cytokines, cytokine variants, analogs, as lacking a comparable effect from a quantitative perspec muteins, and binding compositions derived from antibodies, tive if one agent is only able to achieve 20% of the activity as well as Small molecules. The term also encompasses an 50 of the other agent as determined in an art-accepted assay agent that is neither an agonist nor antagonist, but that can (e.g., a dose-response assay) or in an art-accepted animal bind to a receptor without significantly influencing its bio model. When comparing one result to another result (e.g., logical properties, e.g., signaling or adhesion. Moreover, the one result to a reference standard), “comparable frequently term includes a membrane-bound ligand that has been (though not always) means that one result deviates from a changed by, e.g., chemical or recombinant methods, to a 55 reference standard by less than 35%, by less than 30%, by soluble version of the membrane-bound ligand. A ligand or less than 25%, by less than 20%, by less than 15%, by less receptor may be entirely intracellular, that is, it may reside than 10%, by less than 7%, by less than 5%, by less than 4%. in the cytosol, nucleus, or some other intracellular compart by less than 3%, by less than 2%, or by less than 1%. In ment. The complex of a ligand and receptor is termed a particular embodiments, one result is comparable to a ref “ligand-receptor complex”. 60 erence standard if it deviates by less than 15%, by less than The terms “inhibitors' and “antagonists”, or “activators’ 10%, or by less than 5% from the reference standard. By and "agonists' refer to inhibitory or activating molecules, way of example, but not limitation, the activity or effect may respectively, for example, for the activation of, e.g., a ligand, refer to efficacy, stability, Solubility, or immunogenicity. receptor, cofactor, gene, cell, tissue, or organ. Inhibitors are “Substantially pure' indicates that a component makes up molecules that decrease, block, prevent, delay activation, 65 greater than about 50% of the total content of the compo inactivate, desensitize, or down-regulate, e.g., a gene, pro sition, and typically greater than about 60% of the total tein, ligand, receptor, or cell. Activators are molecules that polypeptide content. More typically, “substantially pure' US 9,598.422 B2 15 16 refers to compositions in which at least 75%, at least 85%, tively, where the change in sequence is artificially intro at least 90% or more of the total composition is the com duced (e.g., muteins); for example, the change is generated ponent of interest. In some cases, the polypeptide will make in the laboratory by human intervention (“hand of man'). up greater than about 90%, or greater than about 95% of the Therefore, non-naturally occurring variants and homologs total content of the composition. may also refer to those that differ from the naturally The terms “specifically binds” or “selectively binds', occurring sequences by one or more conservative Substitu when referring to a ligand/receptor, antibody/antigen, or tions and/or tags and/or conjugates. other binding pair, indicates a binding reaction which is The term “muteins' as used herein refers broadly to determinative of the presence of the protein in a heteroge mutated recombinant proteins. These proteins usually carry neous population of proteins and other biologics. Thus, 10 single or multiple amino acid substitutions and are fre under designated conditions, a specified ligand binds to a quently derived from cloned genes that have been Subjected particular receptor and does not bind in a significant amount to site-directed or random mutagenesis, or from completely to other proteins present in the sample. The antibody, or synthetic genes. binding composition derived from the antigen-binding site The terms "DNA”, “nucleic acid”, “nucleic acid mol of an antibody, of the contemplated method binds to its 15 ecule”, “polynucleotide' and the like are used interchange antigen, or a variant or mutein thereof, with an affinity that ably herein to refer to a polymeric form of nucleotides of any is at least two-fold greater, at least ten times greater, at least length, either deoxyribonucleotides or ribonucleotides, or 20-times greater, or at least 100-times greater than the analogs thereof. Non-limiting examples of polynucleotides affinity with any other antibody, or binding composition include linear and circular nucleic acids, messenger RNA derived therefrom. In a particular embodiment, the antibody (mRNA), complementary DNA (cDNA), recombinant poly will have an affinity that is greater than about 10 liters/mol, nucleotides, vectors, probes, primers and the like. as determined by, e.g., Scatchard analysis (Munsen et al., Indoleamine 2,3-Dioxygenase Analyt. Biochem., 107:220-239 (1980)). As previously alluded to, IDO is an immune regulatory The term “response', for example, of a cell, tissue, organ, enzyme that is normally expressed in tumor cells and in or organism, encompasses a change in biochemical or physi 25 activated immune cells. IDO is one of several immune ological behavior, e.g., concentration, density, adhesion, or response checkpoints that are involved in tumor immune migration within a biological compartment, rate of gene escape; thus, IDO inhibitors disrupt mechanisms by which expression, or state of differentiation, where the change is tumors evade the body's normal immune system. correlated with activation, stimulation, or treatment, or with IDO down-regulates the immune response mediated internal mechanisms such as genetic programming. In cer 30 through oxidation of tryptophan. This results in inhibition of tain contexts, the terms “activation”, “stimulation', and the T-cell activation and induction of T-cell apoptosis, creating like refer to cell activation as regulated by internal mecha an environment in which tumor-specific cytotoxic T lym nisms, as well as by external or environmental factors; phocytes are rendered functionally inactive or are no longer whereas the terms “inhibition”, “down-regulation' and the able to attack a subjects cancer cells. Therefore, therapeutic like refer to the opposite effects. 35 agents aimed at Suppression of tryptophan degradation by The terms “polypeptide”, “peptide', and “protein', used inhibiting IDO activity are desirable. Inhibitors of IDO can interchangeably herein, refer to a polymeric form of amino be used to activate T cells and therefore enhance T cell acids of any length, which can include genetically coded and activation when the T cells are Suppressed by pregnancy, non-genetically coded amino acids, chemically or biochemi malignancy or a virus such as HIV Inhibition of IDO may cally modified or derivatized amino acids, and polypeptides 40 also be an important treatment strategy for patients with having modified polypeptide backbones. The terms include neurological or neuropsychiatric diseases or disorders such fusion proteins, including, but not limited to, fusion proteins as depression. The compounds, compositions and methods with a heterologous amino acid sequence, fusion proteins herein help meet the current need for IDO modulators. with heterologous and homologous leader sequences, with The expression of IDO is modulated by a complex array or without N-terminus methionine residues; immunologi 45 of signals, thus implicating a number of different mecha cally tagged proteins; and the like. nisms of actions. For example, IDO may be induced by As used herein, the terms “variants' and “homologs are inhibition of DNA methyl transferases or histone deacety used interchangeably to refer to amino acid or DNA lases. The NF-KB signaling pathway has also been impli sequences that are similar to reference amino acid or nucleic cated in IDO function. Inhibiting NF-kB activity blocks IDO acid sequences, respectively. The term encompasses natu 50 expression and produces robust anti-tumor responses that rally-occurring variants and non-naturally-occurring vari are both T cell- and IDO-dependent; alternatively, NF-kB ants. Naturally-occurring variants include homologs (poly activation (which may be effected by various factors such as peptides and nucleic acids that differ in amino acid or interferon-YR1/-YR2 signaling and toll-like-receptor activa nucleotide sequence, respectively, from one species to tion) induces IDO gene expression. another), and allelic variants (polypeptides and nucleic acids 55 Other mechanisms are involved with modulation of IDO that differ in amino acid or nucleotide sequence, respec function. By way of example, inhibitors of reactive oxida tively, from one individual to another within a species). tive species (ROS) may effect stabilization of IDO, IDO Thus, variants and homologs encompass naturally occurring levels may be modulated by inhibition or activation of DNA sequences and proteins encoded thereby and their pathways that are both downstream and upstream of IDO; isoforms, as well as splice variants of a protein or gene. The 60 and activation of interferon-Y can activate an autocrine terms also encompass nucleic acid sequences that vary in induction of IDO. one or more bases from a naturally-occurring DNA sequence Studies indicate that the IDO pathway is active in many but still translate into an amino acid sequence that corre cancers, both within tumor cells as a direct defense against sponds to the naturally-occurring protein due to degeneracy T cell attack, and also within antigen-presenting cells of the genetic code. Non-naturally-occurring variants and 65 (APCs) in tumor-draining lymph nodes resulting in periph homologs include polypeptides and nucleic acids that com eral tolerance to tumor-associated antigens (TAAS). Cancers prise a change in amino acid or nucleotide sequence, respec may use the IDO pathway to facilitate survival, growth, US 9,598.422 B2 17 18 invasion, and metastasis of malignant cells expressing TAAS that might otherwise be recognized and attacked by the (I) immune system. As alluded to herein, tryptophan catabolism in tumor tissue by the rate-limiting enzyme IDO provides an oppor tunity for the use of IDO inhibitors as a therapeutic alter native to, or an additive with, conventional chemotherapy. However, certain cancers are capable of catabolizing tryp tophan but are largely IDO-negative. Recent studies indicate that the alternative enzymatic pathway of tryptophan catabo lism involving tryptophan-2,3-dioxygenase (TDO) is also 10 relevant in cancer. TDO, which is considered responsible for regulating systemic tryptophan levels in the liver, is consti or a pharmaceutically acceptable salt, hydrate or Solvate tutively expressed in Some cancers and is also capable of thereof, wherein, Suppressing antitumor immune responses (See, e.g., Platten, M. et al., Cancer Res., 72(21):5435-5440 (Nov. 1, 2012)). 15 the subscript n is 1 or 0; IDO is expressed in a wide variety of human tumors and A is C(O)—, NH SO , CH2—, or tumor cell lines as well as in host APCs, which correlates CHR ; with a worse clinical prognosis. Therefore, inhibition of IDO may improve survival in cancer patients with IDO-mediated B is a bond, —C(O) , NH , —CH2—, or —CHR : immunosuppression. In comparison, TDO is expressed in a T is a bond, CH —NH , O—, OCH , wide variety of human tumors and tumor cell lines, and —C(O)CH , or - CRR : expression of TDO is evident in advanced human glioblas tomas. The identification of tumors expressing high levels of wherein when A is —NH and B is —C(O)—, then T is IDO or TDO may allow more selective inhibition of the other than - C(R)(R') : tryptophan-regulated immunosuppressive pathways. Alter 25 D is N or C(R): natively, compounds inhibiting both IDO and TDO could E is N or C(R): provide the greatest coverage to prevent tumor escape by compensatory expression of the other tryptophan-degrading V is a bond, O , or C(R): enzyme. Therefore, the use of dual IDO/TDO inhibitors or G is an optionally substituted aryl, optionally substituted combinations of IDO- and TDO-specific inhibitors may 30 heteroaryl, or an optionally substituted 9- or 10-mem prove to be a Superior treatment alternative in immuno bered fused bicyclic heteroaryl; therapy of cancer to block immunosuppression mediated by J" is CH, N or C(R), when R is attached to the ring vertex tryptophan metabolism. Although a precise understanding of the underlying identified as J'; mechanism of action by which the compounds of the present 35 R" and R are independently hydrogen, halogen, optionally invention effect their activity is not required to practice the Substituted C-C haloalkyl, optionally Substituted C-C, invention, the compounds (or a subset thereof) are believed cycloalkyl, optionally substituted 3- to 6-membered to inhibit IDO function. Alternatively, the compounds (or a cycloheteroalkyl, optionally substituted phenyl, option subset thereof) may inhibit TDO function. The compounds ally substituted heteroaryl, optionally substituted C-C, (or a subset thereof) may also have inhibitory activity on 40 alkyl, optionally substituted C-C alkoxy, CN, SONH, both IDO and TDO function. Although the compounds of NHSOCH, NHSOCF. OCF, SOCH, SOCF, or the invention are generally referred to herein as IDO inhibi CONH2, and when R' and Rare on adjacent vertices of tors, it is to be understood that the term “IDO inhibitors' a phenyl ring they may be joined together to form a 5- or encompasses compounds that act individually through inhi 6-membered cycloheteroalkyl ring having one or two ring bition of TDO or IDO, and/or compounds that act through 45 vertices independently selected from O, N and S, wherein inhibition of both IDO and TDO. said cycloheteroalkyl ring is optionally substituted with Identification of IDO Inhibitors Possessing Desirable Char from one to three members selected from fluoro and acteristics C-C alkyl: The present invention is drawn, in part, to the identifica tion of inhibitors of IDO with at least one property or 50 R and R' are independently hydrogen, optionally substi characteristic that is of therapeutic relevance. Candidate tuted C-C alkyl, optionally substituted C-C haloalkyl, inhibitors may be identified by using, for example, an fluorine, OH, CN, COH, C(O)NH2, N(R), optionally art-accepted assay or model, examples of which are substituted —O C-C alkyl, —(CRR), OH, described herein. —(CRR), COH, -(CRR), C(O)NH, After identification, candidate inhibitors can be further 55 (CRR), C(O)NHR', (CRR), N(R), NH evaluated by using techniques that provide data regarding (CRR), COH or NH(CRR), C(O)NH; characteristics of the inhibitors (e.g., pharmacokinetic each R is independently H, F, OH, optionally substituted parameters, means of determining solubility or stability). C-C alkyl or optionally substituted —O-C-C alkyl; Comparisons of the candidate inhibitors to a reference standard (which may the “best-of-class” of current inhibi 60 each R is independently H, or optionally substituted C-C, tors) are indicative of the potential viability of such candi alkyl: dates. R is H, OH, F, optionally substituted C-C alkyl, optionally substituted —O C-C alkyl, or N(R): Compounds of the Invention 65 and each m is independently 1, 2, or 3. As noted above, the present invention provides com In some embodiments, the compounds provided herein pounds represented by formula (I): have the formula (Ia): US 9,598.422 B2 19 20 -continued (Ia) (Id) 5 G OY-|- p -CD-x"
10 In some embodiments, the compounds provided herein have the formula (Ie): In some selected embodiments of formula (Ia), com pounds are provided having formulae (Ia1). (Ia2) or (Ia3): 15 (Ie) (Ia1) RI R2 fixN R3 R4 -2J GN -N Y.
v'Nu?, H O 25 (Ia2) In some selected embodiments of formula (Ie), com fix,A pounds are provided having formulae (Iel): R3 R4 -2J 30 (Ie1) GQ N V )n H O
(Ia3) 35 V- R2.
R3 R4 fix,-2J 40 N G )n H O In some embodiments, the compounds provided herein have the formula (If). (Ig), or (Ih): In some embodiments, the compounds provided herein 45 have the formula (Ib), (Ic), or (Id): (If) (Ib) fixR1 R2 50 -2J \, -O. 1Nur 55 (Ic) (Ig) R1 2 fix 60 R5 -2J
65 US 9,598.422 B2 21 22 -continued Methods of Synthesis (Ih) R The compounds of the present invention may be prepared Ary R2. from starting materials which are known in the chemical fix, literature or are commercially available by methods such as R5 -2J those illustrated in the following Schemes utilizing chemical H transformations known to those skilled in the art of organic G N chemistry. Solvents, temperatures, pressures, and other reac Yy -E )n R3 tion conditions may readily be selected by one of ordinary O 10 skill in the art. These Schemes are illustrative and are not meant to limit the possible techniques one skilled in the art In some embodiments, the compounds provided herein may use to manufacture compounds disclosed herein. Dif have the formula (Ii) or (I): ferent methods may be evident to those skilled in the art. 15 Additionally, the various steps in the synthesis may be performed in an alternate sequence or order to give the (II) desired compound(s). Further, the representation of the reactions in these Schemes as discrete steps does not pre clude their being performed in tandem, either by telescoping multiple steps in the same reaction vessel or by performing multiple steps without purifying or characterizing the inter mediate(s). In addition, many of the compounds prepared by O's the methods below can be further modified using conven (I) tional chemistry well known to those skilled in the art. All 25 documents cited herein are incorporated herein by reference G ?\, e x in their entirety. References to many of these chemical transformations N-'Ur N-K) employed herein can be found in Smith, M. B. et al., March's Advanced Organic Chemistry Reactions, Mecha 30 nisms, and Structure, Fifth Edition, Wiley-Interscience, New For each of the above formulae (Ia), (Ia1). (Ia2). (Ia3), York (2001), or other standard texts on the topic of synthetic (Ib), (Ic), (Id). (Ie), (Ie1). (If). (Ig), (Ih), (Ii) and (I), each of organic chemistry. Certain transformations may require that the subscript, letters, J. R. R. R. R. and R have the reactive functional groups be masked by protecting group meanings provided with reference to formula (I), unless 35 (s). A convenient reference which provides conditions for noted otherwise. introduction, removal, and relative Susceptibility to reaction In one group of selected embodiments, any one com conditions of these groups is Greene, T. W. et al., Protective pound of FIG. 1 is provided. Groups in Organic Synthesis. Third Edition, Wiley-Inter In another group of selected embodiments, any one com science, New York (1999). pound of FIG. 1 is provided having an activity level iden 40 tified as “A” or “B”. Scheme 1 In another group of selected embodiments, any one com pound of FIG. 1 is provided having an activity level iden Reverse Amides and Sulfonamides, Cycloalkyl tified as “A. Core, Direct or O-Linked G
Scheme 1
O O O 1.) MH, R'n' O )n OR OR III PhN(Tf) -- 2.) H2, catalyst )n base )n 3.) aq. HX O O
O OTf II IV V NaBH4 1.) G-B(OR)2 (V = a bond, Pd', base E = CH) US 9,598.422 B2
-continued
(I) O
C R3 A1 OR J r )n o R1
NaR2 base, GX O XVIa OH (V = O, E = CH) VII GOH, DIAD Ph3P
O O R3 NH2 R3 R3 OH OR R4 R4 )n DPPA, RN )n 1.) LiHMDS, RX )n re A 2.) LiOH, water E then aq. E E LiOH V.No. V.No. W.No. IX VIII VI
Treatment of a phosphonoacetate ester (III), with a base 35 structure VII. These alcohols can be deprotonated with bases such as sodium hydride in a solvent such as THF (Scheme such as sodium hydride or KHMDS, and the resulting 1) followed by a ketone of the general structure II affords a alkoxides can react with aryl or heteroaryl halides under trisubstituted olefin. Substituted analogs of III (R is not H) SNAr conditions to afford additional intermediates VI afford tetrasubstituted olefins. This method and additional (where V-O and E=CH). Alternatively, reaction can be 40 accomplished by activation of alcohol VII with DIAD. methods described below are transformations familiar to DEAD, or a related azodicarboxylate and a trialkyl or those skilled in the art of organic/medicinal chemistry. triarylphosphine and coupling with a phenol or related Alternative methods for olefination and the transformations heteroaryl GOH. (Mitsunobu reaction) Intermediates VI described below are known and will be selected by one (and later intermediates) may be obtained as mixtures of cis skilled in the art based on their applicability to the specific 45 and trans isomers. Methods for control of the stereochemical Substrate under consideration. Reduction is accomplished by outcome of the above reactions are known to those familiar stirring or shaking a solution of the olefin in a Suitable in the art of organic/medicinal chemistry. Additionally, Solvent under an atmosphere or more of H in the presence methods for the separation of these isomers are known and of a catalyst, normally palladium on carbon. Hydrolysis of described in detail in the synthetic examples. If required, the the ketal group affords a ketone of the general structure IV. 50 group R' can be appended by alkylation of intermediate VI. Typically, this is accomplished by heating with an aqueous Methods for control of the absolute stereochemistry of the acid such as HCl in the presence of a co-solvent Such as resulting asymmetric center are known to those familiar with THF. In addition to the cyclic ethylene glycol-based ketal the art, as are chiral separation methods. Saponification of shown, other cyclic and acyclic ketal protecting groups the ester by heating with aq. LiOH or a similar base, could be used. Ketones are deprotonated with bases such as 55 generally in the presence of an organic co-solvent such as LiHMDS and react with N-phenyltrifluoromethanesulfon THF affords carboxylic acids VIII. Acids VIII can be rear imide or similar reagents to afford triflates of the general ranged, usually by heating with DPPA and triethylamine structure V. These triflates participate in Suzuki couplings (Curtius and related rearrangements), and the intermediate (T. Ishiyama, M. Murata, N. Miyaura, J. Org. Chem., 1995, isocyanates react with aq. base to afford primary amines IX. 60, 7508-7510) with boronic acids or esters G-B(OR), to 60 These can react with electrophiles including, but not limited afford coupled products. Many variations on this reaction are to acid chlorides and sulfonyl chlorides XVIa to afford known, but generally it involves heating the two Substrates compounds of the invention I. Another means of preparing and a catalyst such as (Ph-P), Pd in a solvent such as DMF compounds I from IX where A is CO uses the carboxylic with a base such as aq. potassium carbonate. Reduction of acid derivative of XVIa and peptide coupling reagents. For the olefin provides intermediate VI (where V—a bond and 65 a recent review of peptide coupling methods see: Ayman E=CH). Ketones IV can be reduced by NaBH or similar El-Faham and Fernando Albericio. Chem. Rev. 2011, 111, hydride reducing agents to afford alcohols of the general 6557-66O2. US 9,598.422 B2 25 Scheme 2 -continued
O Reverse Amides and Sulfonamides, Piperidine Core, Direct or C-Linked G O W = a bond: Hipbase, GX )n )n V = C(CR): base, G(CR)2X E Ho Scheme 2 N W. 10 No.
XIII XII OR (E = N) OR GX, base He (V = a bond) )n 15
Her Scheme 3 illustrates a method of making intermediate VI (V = C(R).) E by performing the steps of Scheme 1 in a different order. N Intermediate II can be converted to a triflate as described above and coupled with a boronic acid or ester G-B(OR), to give intermediate XI (V—a bond). Analogs where V=O can (E = N) be prepared by reduction of ketone II and conversion to the ether XI as described above. Ketal hydrolysis affords ketone Piperidine and pyrrolidine esters X are known compounds 25 XII. Transformation to intermediate VI is accomplished by and can undergo S.Ar (V—a bond) and N-alklyation (V-C olefination and reduction as described above. Intermediates (R)) reactions to afford intermediates VI (E-N). These XII in which E=N can be prepared from ketones XIII using intermediates may be transformed to compounds of the SNAr (V—a bond) or alkylation (V=C(R).) chemistry. invention I as shown in Scheme I. 30
Scheme 3 Scheme 4
Reverse Amides and Sulfonamides, Cycloalkyl or 35 Piperidine Core, Direct C, or O-Linked G. Truncated Normal Amides, Cycloalkyl or Alternate Method Piperidine Core, Direct, C, or O-Linked G
40 Scheme 3 Scheme 4 7 \ W = a bond / \ O O 1.) base, ArN(Tf)2 O O O CO2H )n 2.)) Suzuki couplingli )n ketal 45 )n )n Bop or other W = O - -> 1.) TosMIC coupling agent 1.) NaBH4 hydrolysis He- I E 2.) H3O+ NH2 2.) GX (SNAr) or O GOH (Mitsunobu) VQ, 50 G VQ, G Van G J 1. HR XI XII XIV Y al II (E = CH) 2 O O XVIb R 55 OR (T = a bond, B = CO, A = NH) )n 1.) olefination -- 2.) reduction n 60 V. E No. Scheme 4 illustrates a method of making additional V. compounds of the invention. Ketone XII can be transformed No. into a nitrile by the use of TosMIC (Van Leusen reaction). XII VI 65 Hydrolysis of the nitrile affords acid XIV which can be converted into compounds of the invention I by treatment with amines under amide coupling conditions. US 9,598.422 B2 27 28 Scheme 5 -continued
Ureas and Phenylaceticamides of Cyclohexylamine or 4-aminopiperidine, Direct, C, or O-Linked G OH
n 1.) RCOCl, Et3N Scheme 5 Hip 2.) n-BuLi O NH2 10 O
)n )n HN -( NHOAc -- I NaCNBH NCO XVIINo. R --/O E ROH E 1s 15 XVIII W. V. 1N nG nG J R1 O XII XV ex O R2 O XVIc N
Bop, Et3N, DMF NaHMDS XV I R -e- HOC R3 )n THF then R-X 25 1N -HR VQ, NeaR2 XVId 30
Scheme 5 illustrates a method of making additional compounds of the invention. Ketones XII can be trans R N R formed into primary amines XV by reductive amination with 35 OH LiOH ammonia or an ammonium salt. This reaction is generally R He performed using Sodium cyanoborohydride in an alcoholic n H2O2 )n solvent. Treatment with isocyanates XVIc affords ureas which are compounds of the invention I. Arylacetamides which are compounds of the invention I are obtained by 40 coupling amine XV with arylacetic acids XVId using a V. V. reagent Such as Bop and a tertiary amine base in a solvent No. No. Such as DMF. XX VIII
45 Scheme 6 Scheme 6 illustrates a method for controlling the absolute Control of Absolute Stereochemistry in the stereochemistry of intermediate VIII and materials arising Conversion of VI to VIII 50 from it. Saponification of esters VI provides carboxylic acids XVII. Treatment of these acids with an acid chloride such as pivaloyl chloride provides a mixed anhydride intermediate. Scheme 6 In a separate vessel, an optically pure oxazolidinone of O known stereochemistry and general structure XVIII is 55 deprotonated by treatment with a strong base Such as OR n-BuLi. These activated species are combined to form the acyloxazolidinone XIX which is deprotonated by bases such as NaHMDS. Alkylation of the resulting enolate proceeds )n LiOH 60 with predictable control of stereochemistry at the newly THF, water formed center to provide materials XX. Removal of the chiral auxiliary to give optically-active carboxylic acids VIII W.No. is accomplished by treatment with a solution of basic 65 hydrogen peroxide. For a review of the history and scope VI scope of this reaction see: D. A. Evans, M. D. Ennis, D. J. Mathre. J. Am. Chem. Soc., 1982, 104 (6), pp 1737-1739. US 9,598.422 B2 29 30 Scheme 7 -continued Synthesis of Compounds of the Invention (I) where RO R -OH HO
)n R- (I) Scheme 7 O O 10 W. R R No. OR OR XXIII G-Li Ho- He- I) )n (V = a bond) )n Ps ( 15 As shown in Scheme 8, a ketone of general structure XII can be treated with a halo acetate where X=Br in the presence of Zinc metal (Reformatsky reaction) to give the a O HO. G. tertiary alcohol of general structure XXIII. The ester XXIII can be converted to a compound of the Invention (I) by IV XXI methods already described herein. In addition to the above general schemes, the compounds MgBr-V-G described herein can be prepared by representative methods (V = CH) as provided in the Examples below. Modifications to Enhance Inhibitor Characteristics O 25 It is frequently beneficial, and sometimes imperative, to R3 improve one of more physical properties of the treatment OR modalities disclosed herein and/or the manner in which they are administered. Improvements of physical properties )n - (I), include, for example, methods of increasing water Solubility, 30 bioavailability, serum half-life, and/or therapeutic half-life; and/or modulating biological activity. Modifications known in the art include pegylation, Fc HO W fusion and albumin fusion. Although generally associated with large molecule agents (e.g., polypeptides), such modi G 35 fications have recently been evaluated with particular small XXII molecules. By way of example, Chiang, M. et al., (J. Am. Chem. Soc., 136(9):3370-3373 (2014)) describe a small As shown in Scheme 7, a ketone of general structure IV molecule agonist of the adenosine 2a receptor conjugated to can be treated with a the lithiate G-Li, which can be the immunoglobulin Fc domain. The small molecule-Fc generated by several methods well-known to one skilled in 40 conjugate retained potent Fc receptor and adenosine 2a the art, to produce a tertiary alcohol of general structure receptor interactions and showed Superior properties com XXI. The ester of general structure XXXI can be converted pared to the unconjugated Small molecule. Covalent attach to a compound of general structure I via methods already ment of PEG molecules to small molecule therapeutics has described herein. Alternatively, the ketone IV can be treated also been described (Li, W. et al., Progress in Polymer 45 Science, 38:421-444 (2013)). with the organometallic MgBr V-G (Grignard reagent) to Therapeutic and Prophylactic Uses give a tertiary alcohol of general structure XXII. The present invention contemplates the use of the TDO Scheme 8 inhibitors described herein in the treatment or prevention of a broad range of diseases, disorders and/or conditions, Synthesis of Compounds of the Invention (I) where 50 and/or the symptoms thereof. While particular uses are R—OH described in detail hereafter, it is to be understood that the present invention is not so limited. Furthermore, although general categories of particular diseases, disorders and con ditions are set forth hereafter, some of the diseases, disorders Scheme 8 55 and conditions may be a member of more than one category, and others may not be a member of any of the disclosed O O categories. R Oncology-Related Disorders. OR In accordance with the present invention, an IDO inhibi )n 60 tor can be used to treat or prevent a proliferative condition - A - or disorder, including a cancer, for example, cancer of the E Zn uterus, cervix, breast, prostate, testes, gastrointestinal tract (e.g., esophagus, oropharynx, Stomach, Small or large intes W. No. tines, colon, or rectum), kidney, renal cell, bladder, bone, 65 bone marrow, skin, head or neck, liver, gallbladder, heart, XII lung, pancreas, salivary gland, adrenal gland, thyroid, brain (e.g., gliomas), ganglia, central nervous system (CNS) and US 9,598.422 B2 31 32 peripheral nervous system (PNS), and cancers of the Rheumatoid Arthritis (RA), which is generally character hematopoietic system and the immune system (e.g., spleen ized by chronic inflammation in the membrane lining (the or thymus). The present invention also provides methods of synovium) of the joints, affects approximately 1% of the treating or preventing other cancer-related diseases, disor U.S. population (-2.1 million people). Further understand ders or conditions, including, for example, immunogenic ing of the role of cytokines, including TNF-C. and IL-1, in tumors, non-immunogenic tumors, dormant tumors, virus the inflammatory process has enabled the development and induced cancers (e.g., epithelial cell cancers, endothelial cell introduction of a new class of disease-modifying antirheu cancers, squamous cell carcinomas and papillomavirus), matic drugs (DMARDs). Agents (some of which overlap adenocarcinomas, lymphomas, carcinomas, melanomas, with treatment modalities for RA) include ENBREL(R) (etan leukemias, myelomas, sarcomas, teratocarcinomas, chemi 10 ercept), REMICADE(R) (infliximab), HUMIRAR) (adalim cally-induced cancers, metastasis, and angiogenesis. The umab) and KINERETR (anakinra). Though some of these invention contemplates reducing tolerance to a tumor cell or agents relieve symptoms, inhibit progression of structural cancer cell antigen, e.g., by modulating activity of a regu damage, and improve physical function in particular patient latory T-cell and/or a CD8+ T-cell (see, e.g., Ramirez populations, there is still a need for alternative agents with Montagut et al., Oncogene, 22:3180-3 187 (2003); and 15 improved efficacy, complementary mechanisms of action, Sawaya et al., New Engl. J. Med., 349:1501-1509 (2003)). In and fewer/less severe adverse effects. particular embodiments, the tumor or cancer is colon cancer, Psoriasis, a constellation of common immune-mediated ovarian cancer, breast cancer, melanoma, lung cancer, glio chronic skin diseases, affects more than 4.5 million people blastoma, or leukemia. The use of the term(s) cancer-related in the U.S., of which 1.5 million are considered to have a diseases, disorders and conditions is meant to refer broadly moderate-to severe form of the disease. Moreover, over 10% to conditions that are associated, directly or indirectly, with of patients with psoriasis develop psoriatic arthritis, which cancer, and includes, e.g., angiogenesis and precancerous damages the bone and connective tissue around the joints. conditions such as dysplasia. An improved understanding of the underlying physiology of In Some embodiments, the present invention provides psoriasis has resulted in the introduction of agents that, for methods for treating a proliferative condition, cancer, tumor, 25 example, target the activity of T lymphocytes and cytokines or precancerous condition with an IDO inhibitor and at least responsible for the inflammatory nature of the disease. Such one additional therapeutic or diagnostic agent, examples of agents include the TNF-C. inhibitors (also used in the treat which are set forth elsewhere herein. ment of rheumatoid arthritis (RA)), including ENBREL(R) Immune- and Inflammatory-Related Disorders. (etanercept), REMICADE(R) (infliximab) and HUMIRAR As used herein, terms such as “immune disease'. 30 (adalimumab)), and T-cell inhibitors such as AMEVIVER “immune condition”, “immune disorder”, “inflammatory (alefacept) and RAPTIVAR (efalizumab). Though several of disease”, “inflammatory condition”, “inflammatory disor these agents are effective to some extent in certain patient der” and the like are meant to broadly encompass any populations, none have been shown to effectively treat all immune- or inflammatory-related condition (e.g., pathologi patients. cal inflammation and autoimmune diseases). Such condi 35 Subjects suffering from multiple sclerosis (MS), a seri tions frequently are inextricably intertwined with other ously debilitating autoimmune disease comprising multiple diseases, disorders and conditions. By way of example, an areas of inflammation and scarring of the myelin in the brain “immune condition” may refer to proliferative conditions, and spinal cord, may be particularly helped by the IDO Such as cancer, tumors, and angiogenesis; including infec inhibitors described herein, as current treatments only alle tions (acute and chronic), tumors, and cancers that resist 40 viate symptoms or delay the progression of disability. eradication by the immune system. Similarly, the IDO inhibitors may be particularly advan A non-limiting list of immune- and inflammatory-related tageous for Subjects afflicted with neurodegenerative disor diseases, disorders and conditions which may be treated or ders, such as Alzheimer's disease (AD), a brain disorder that prevented with the compounds and compositions of the seriously impairs patients thought, memory, and language present invention include, arthritis (e.g., rheumatoid arthri 45 processes; and Parkinson's disease (PD), a progressive dis tis), kidney failure, lupus, asthma, psoriasis, colitis, pan order of the CNS characterized by, for example, abnormal creatitis, allergies, fibrosis, Surgical complications (e.g., movement, rigidity and tremor. These disorders are progres where inflammatory cytokines prevent healing), anemia, and sive and debilitating, and no curative agents are available. fibromyalgia. Other diseases and disorders which may be Viral-Related Disorders. associated with chronic inflammation include Alzheimer's 50 The present invention contemplates the use of the IDO disease, congestive heart failure, stroke, aortic valve Steno inhibitors in the treatment and/or prevention of any viral sis, arteriosclerosis, osteoporosis, Parkinson's disease, disease, disorder or condition for which treatment with an infections, inflammatory bowel disease (e.g., Crohn's dis IDO inhibitor may be beneficial. In particular embodiments, ease and ulcerative colitis), allergic contact dermatitis and the viral disorder is a chronic viral disorder. Examples of other eczemas, systemic Sclerosis, transplantation and mul 55 viral diseases, disorders and conditions that are contem tiple Sclerosis. plated include, but are not limited to, hepatitis B virus Among other immune-related disorders, it is contem (HBV), hepatitis C virus (HCV), human papilloma virus plated that inhibition of IDO function may also play a role (HPV), HIV, AIDS (including its manifestations such as in immunologic tolerance and prevention offetal rejection in cachexia, dementia, and diarrhea), herpes simplex virus uterO. 60 (HSV), Epstein-Barr virus (EBV), varicella Zoster virus, In some embodiments, an IDO inhibitor described herein coxsackie virus, and cytomegalovirus (CMV). can be combined with an immunosuppressive agent to Bacterial- and Parasitic-Related Disorders. reduce the number of immune effector cells. Embodiments of the present invention contemplate the Some of the aforementioned diseases, disorders and con administration of the IDO inhibitors described herein to a ditions for which an IDO inhibitor may be particularly 65 subject for the treatment of a bacterial infection, for efficacious (due to, for example, limitations of current thera example, a Mycobacterium infection (e.g., Mycobacterium pies) are described in more detail hereafter. leprae or Mycobacterium tuberculosis) or an infection US 9,598.422 B2 33 34 caused by Listeria monocytogenes or Toxplasma gondii. ethylene-vinylacetate, methylcellulose, carboxymethylcel Other embodiments contemplate the treatment of a parasitic lulose, protamine Sulfate, or lactide/glycolide copolymers, infection including, but not limited to, Leishmania dono polylactide/glycolide copolymers, or ethylenevinylacetate vani, Leishmania tropica, Leishmania major; Leishmania copolymers in order to control delivery of an administered aethiopica, Leishmania mexicana, Plasmodium falciparum, composition. For example, the oral agent can be entrapped Plasmodium vivax, Plasmodium ovale, or Plasmodium in microcapsules prepared by coacervation techniques or by malariae. Frequently, anti-parasitic therapy is administered interfacial polymerization, by the use of hydroxymethylcel prophylactically (e.g., before a Subject travels to an area with lulose or gelatin-microcapsules or poly(methylmethacro a high frequency of parasitic infection). late) microcapsules, respectively, or in a colloid drug deliv 10 ery system. Colloidal dispersion systems include Pharmaceutical Compositions macromolecule complexes, nano-capsules, microspheres, The IDO inhibitors of the present invention may be in the microbeads, and lipid-based systems, including oil-in-water form of compositions suitable for administration to a Sub emulsions, micelles, mixed micelles, and liposomes. Meth ject. In general. Such compositions are “pharmaceutical 15 ods for the preparation of the above-mentioned formulations compositions' comprising an IDO inhibitor(s) and one or will be apparent to those skilled in the art. more pharmaceutically acceptable or physiologically Formulations for oral use may also be presented as hard acceptable diluents, carriers or excipients. In certain gelatin capsules wherein the active ingredient is mixed with embodiments, the IDO inhibitors are present in a therapeu an inert Solid diluent, for example, calcium carbonate, tically acceptable amount. The pharmaceutical compositions calcium phosphate, kaolin or microcrystalline cellulose, or may be used in the methods of the present invention; thus, as Soft gelatin capsules wherein the active ingredient is for example, the pharmaceutical compositions can be mixed with water or an oil medium, for example, peanut oil, administered ex vivo or in vivo to a subject in order to liquid paraffin, or olive oil. practice the therapeutic and prophylactic methods and uses Aqueous Suspensions contain the active materials in described herein. 25 admixture with excipients suitable for the manufacture The pharmaceutical compositions of the present invention thereof. Such excipients can be suspending agents, for can be formulated to be compatible with the intended example, sodium carboxymethylcellulose, methylcellulose, method or route of administration; exemplary routes of hydroxy-propylmethylcellulose, Sodium alginate, polyvinyl administration are set forth herein. Furthermore, the phar pyrrollidone, gum tragacanth and gum acacia; dispersing or maceutical compositions may be used in combination with 30 wetting agents, for example, a naturally-occurring phospha other therapeutically active agents or compounds as tide (e.g., lecithin), or condensation products of an alkylene described herein in order to treat or prevent the diseases, oxide with fatty acids (e.g., polyoxyethylene stearate), or disorders and conditions as contemplated by the present condensation products of ethylene oxide with long chain invention. aliphatic alcohols (e.g., for heptadecaethyleneoxycetanol), The pharmaceutical compositions containing the active 35 or condensation products of ethylene oxide with partial ingredient (e.g., an inhibitor of IDO function) may be in a esters derived from fatty acids and a hexitol (e.g., polyoxy form Suitable for oral use, for example, as tablets, capsules, ethylene Sorbitol monooleate), or condensation products of troches, lozenges, aqueous or oily suspensions, dispersible ethylene oxide with partial esters derived from fatty acids powders or granules, emulsions, hard or soft capsules, or and hexitol anhydrides (e.g., polyethylene Sorbitan syrups, solutions, microbeads or elixirs. Pharmaceutical 40 monooleate). The aqueous Suspensions may also contain one compositions intended for oral use may be prepared accord or more preservatives. ing to any method known to the art for the manufacture of Oily Suspensions may be formulated by Suspending the pharmaceutical compositions, and Such compositions may active ingredient in a vegetable oil, for example, arachis oil, contain one or more agents such as, for example, Sweetening olive oil, sesame oil or coconut oil, or in a mineral oil Such agents, flavoring agents, coloring agents and preserving 45 as liquid paraffin. The oily Suspensions may contain a agents in order to provide pharmaceutically elegant and thickening agent, for example, beeswax, hard paraffin or palatable preparations. Tablets, capsules and the like contain cetyl alcohol. Sweetening agents such as those set forth the active ingredient in admixture with non-toxic pharma above, and flavoring agents may be added to provide a ceutically acceptable excipients which are suitable for the palatable oral preparation. manufacture of tablets. These excipients may be, for 50 Dispersible powders and granules Suitable for preparation example, diluents, such as calcium carbonate, sodium car of an aqueous Suspension by the addition of water provide bonate, lactose, calcium phosphate or Sodium phosphate: the active ingredient in admixture with a dispersing or granulating and disintegrating agents, for example, corn wetting agent, Suspending agent and one or more preserva starch, or alginic acid; binding agents, for example, starch, tives. Suitable dispersing or wetting agents and Suspending gelatin or acacia, and lubricating agents, for example, mag 55 agents are exemplified herein. nesium Stearate, Stearic acid or talc. The pharmaceutical compositions of the present invention The tablets, capsules and the like suitable for oral admin may also be in the form of oil-in-water emulsions. The oily istration may be uncoated or coated by known techniques to phase may be a vegetable oil, for example, olive oil or delay disintegration and absorption in the gastrointestinal arachis oil, or a mineral oil, for example, liquid paraffin, or tract and thereby provide a Sustained action. For example, a 60 mixtures of these. Suitable emulsifying agents may be time-delay material Such as glyceryl monostearate or glyc naturally occurring gums, for example, gum acacia or gum eryl distearate may be employed. They may also be coated tragacanth; naturally occurring phosphatides, for example, by techniques known in the art to form osmotic therapeutic Soybean, lecithin, and esters or partial esters derived from tablets for controlled release. Additional agents include fatty acids; hexitol anhydrides, for example, Sorbitan biodegradable or biocompatible particles or a polymeric 65 monooleate; and condensation products of partial esters with Substance such as polyesters, polyamine acids, hydrogel, ethylene oxide, for example, polyoxyethylene Sorbitan polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid, monooleate. US 9,598.422 B2 35 36 Formulations can also include carriers to protect the Suspension in a non-toxic parenterally-acceptable diluent or composition against rapid degradation or elimination from Solvent, for example, as a solution in 1,3-butane diol. the body, such as a controlled release formulation, including Acceptable diluents, solvents and dispersion media that may implants, liposomes, hydrogels, prodrugs and microencap be employed include water, Ringer's Solution, isotonic Sulated delivery systems. For example, a time delay material 5 sodium chloride solution, CREMOPHORR EL (BASF, Par Such as glyceryl monostearate or glyceryl Stearate alone, or sippany, N.J.) or phosphate buffered saline (PBS), ethanol, in combination with a wax, may be employed. polyol (e.g., glycerol, propylene glycol, and liquid polyeth The pharmaceutical compositions typically comprise a ylene glycol), and Suitable mixtures thereof. In addition, therapeutically effective amount of an IDO inhibitor con sterile, fixed oils are conventionally employed as a solvent templated by the present invention and one or more phar 10 maceutically and physiologically acceptable formulation or Suspending medium. For this purpose any bland fixed oil agents. Suitable pharmaceutically acceptable or physiologi may be employed, including synthetic mono- or diglycer cally acceptable diluents, carriers or excipients include, but ides. Moreover, fatty acids such as oleic acid, find use in the are not limited to, antioxidants (e.g., ascorbic acid and preparation of injectables. Prolonged absorption of particu Sodium bisulfate), preservatives (e.g., benzyl alcohol, 15 lar injectable formulations can be achieved by including an methyl parabens, ethyl or n-propyl, p-hydroxybenzoate), agent that delays absorption (e.g., aluminum monostearate emulsifying agents, Suspending agents, dispersing agents, or gelatin). Solvents, fillers, bulking agents, detergents, buffers, vehicles, The present invention contemplates the administration of diluents, and/or adjuvants. For example, a Suitable vehicle the IDO inhibitors in the form of suppositories for rectal may be physiological saline solution or citrate buffered administration. The Suppositories can be prepared by mixing saline, possibly Supplemented with other materials common the drug with a suitable non-irritating excipient which is in pharmaceutical compositions for parenteral administra Solid at ordinary temperatures but liquid at the rectal tem tion. Neutral buffered saline or saline mixed with serum perature and will therefore melt in the rectum to release the albumin are further exemplary vehicles. Those skilled in the drug. Such materials include, but are not limited to, cocoa art will readily recognize a variety of buffers that can be used 25 butter and polyethylene glycols. in the pharmaceutical compositions and dosage forms con The IDO inhibitors contemplated by the present invention templated herein. Typical buffers include, but are not limited may be in the form of any other suitable pharmaceutical to, pharmaceutically acceptable weak acids, weak bases, or composition (e.g., sprays for nasal or inhalation use) cur mixtures thereof. As an example, the buffer components can rently known or developed in the future. be water soluble materials such as phosphoric acid, tartaric 30 The concentration of a polypeptide or fragment thereof in acids, lactic acid, Succinic acid, citric acid, acetic acid, a formulation can vary widely (e.g., from less than about ascorbic acid, aspartic acid, glutamic acid, and salts thereof. 0.1%, usually at or at least about 2% to as much as 20% to Acceptable buffering agents include, for example, a Tris 50% or more by weight) and will usually be selected buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic primarily based on fluid volumes, viscosities, and Subject acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid 35 based factors in accordance with, for example, the particular (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt mode of administration selected. (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), Routes of Administration and N-tris hydroxymethylmethyl-3-aminopropanesulfonic The present invention contemplates the administration of acid (TAPS). IDO inhibitors, and compositions thereof, in any appropriate After a pharmaceutical composition has been formulated, 40 manner. Suitable routes of administration include oral, par it may be stored in sterile vials as a Solution, Suspension, gel. enteral (e.g., intramuscular, intravenous, Subcutaneous (e.g., emulsion, solid, or dehydrated or lyophilized powder. Such injection or implant), intraperitoneal, intracisternal, intraar formulations may be stored either in a ready-to-use form, a ticular, intraperitoneal, intracerebral (intraparenchymal) and lyophilized form requiring reconstitution prior to use, a intracerebroventricular), nasal, vaginal, Sublingual, liquid form requiring dilution prior to use, or other accept 45 intraocular, rectal, topical (e.g., transdermal). Sublingual and able form. In some embodiments, the pharmaceutical com inhalation. Depot injections, which are generally adminis position is provided in a single-use container (e.g., a single tered subcutaneously or intramuscularly, may also be uti use vial, ampoule, Syringe, or autoinjector (similar to, e.g., lized to release the IDO inhibitors disclosed herein over a an EPIPENR)), whereas a multi-use container (e.g., a multi defined period of time. use vial) is provided in other embodiments. Any drug 50 Particular embodiments of the present invention contem delivery apparatus may be used to deliver and IDO inhibitor, plate oral administration. including implants (e.g., implantable pumps) and catheter Combination Therapy systems, slow injection pumps and devices, all of which are The present invention contemplates the use of IDO inhibi well known to the skilled artisan. Depot injections, which tors in combination with one or more active therapeutic are generally administered Subcutaneously or intramuscu 55 agents (e.g., chemotherapeutic agents) or other prophylactic larly, may also be utilized to release the polypeptides dis or therapeutic modalities (e.g., radiation). In Such combina closed herein over a defined period of time. Depot injections tion therapy, the various active agents frequently have are usually either solid- or oil-based and generally comprise different, complementary mechanisms of action. Such com at least one of the formulation components set forth herein. bination therapy may be especially advantageous by allow One of ordinary skill in the art is familiar with possible 60 ing a dose reduction of one or more of the agents, thereby formulations and uses of depot injections. reducing or eliminating the adverse effects associated with The pharmaceutical compositions may be in the form of one or more of the agents. Furthermore, Such combination a sterile injectable aqueous or oleagenous Suspension. This therapy may have a synergistic therapeutic or prophylactic Suspension may be formulated according to the known art effect on the underlying disease, disorder, or condition. using those Suitable dispersing or wetting agents and Sus 65 As used herein, "combination' is meant to include thera pending agents mentioned herein. The sterile injectable pies that can be administered separately, for example, for preparation may also be a sterile injectable solution or mulated separately for separate administration (e.g., as may US 9,598.422 B2 37 38 be provided in a kit), and therapies that can be administered thamine oxide hydrochloride, melphalan, novembichin, together in a single formulation (i.e., a “co-formulation'). phenesterine, prednimustine, trofosfamide, uracil mustard; In certain embodiments, the IDO inhibitors are adminis nitroSureas such as carmustine, chlorozotocin, fotemustine, tered or applied sequentially, e.g., where one agent is admin lomustine, nimustine, ranimustine; antibiotics Such as acla istered prior to one or more other agents. In other embodi cinomysins, actinomycin, authramycin, aZaserine, bleomy ments, the IDO inhibitors are administered simultaneously, cins, cactinomycin, calicheamicin, carabicin, caminomycin, e.g., where two or more agents are administered at or about carzinophilin, chromomycins, dactinomycin, daunorubicin, the same time; the two or more agents may be present in two detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epi or more separate formulations or combined into a single rubicin, esorubicin, idarubicin, marcellomycin, mitomycins, formulation (i.e., a co-formulation). Regardless of whether 10 mycophenolic acid, nogalamycin, olivomycins, peplomycin, the two or more agents are administered sequentially or simultaneously, they are considered to be administered in potfiromycin, puromycin, quelamycin, rodorubicin, Strep combination for purposes of the present invention. tonigrin, Streptozocin, tubercidin, ubenimex, Zinostatin, The IDO inhibitors of the present invention may be used Zorubicin; anti-metabolites such as methotrexate and 5-fluo in combination with at least one other (active) agent in any 15 rouracil (5-FU); folic acid analogs such as denopterin, manner appropriate under the circumstances. In one embodi methotrexate, pteropterin, trimetrexate; purine analogs such ment, treatment with the at least one active agent and at least as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; one IDO inhibitor of the present invention is maintained pyrimidine analogs such as ancitabine, azacitidine, 6-azau over a period of time. In another embodiment, treatment ridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, with the at least one active agent is reduced or discontinued enocitabine, floXuridine, 5-FU; androgens such as caluster (e.g., when the Subject is stable), while treatment with an one, dromostanolone propionate, epitioStanol, mepitioStane, IDO inhibitor of the present invention is maintained at a testolactone; anti-adrenals such as aminoglutethimide, mito constant dosing regimen. In a further embodiment, treatment tane, triloStane; folic acid replenisher such as frolinic acid; with the at least one active agent is reduced or discontinued aceglatone; aldophosphamide glycoside; aminolevulinic (e.g., when the Subject is stable), while treatment with an 25 acid; amsacrine; bestrabucil; bisantrene, edatraxate; defo IDO inhibitor of the present invention is reduced (e.g., lower famine; demecolcine; diaziquone; elformithine; elliptinium dose, less frequent dosing or shorter treatment regimen). In acetate; etoglucid: gallium nitrate; hydroxyurea; lentinan; yet another embodiment, treatment with the at least one lonidamine; mitoguaZone; mitoxantrone; mopidamol; nitra active agent is reduced or discontinued (e.g., when the crine; pentostatin: phenamet, pirarubicin; podophyllinic subject is stable), and treatment with the IDO inhibitor of the 30 acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; present invention is increased (e.g., higher dose, more spirogermanium; tenuaZonic acid; triaziquone; 2.2.2"- frequent dosing or longer treatment regimen). In yet another trichlorotriethylamine; urethan; vindesine; dacarbazine: embodiment, treatment with the at least one active agent is mannomustine, mitobronitol; mitolactol; pipobroman; gacy maintained and treatment with the IDO inhibitor of the tosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; present invention is reduced or discontinued (e.g., lower 35 taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gem dose, less frequent dosing or shorter treatment regimen). In citabine; 6-thioguanine; mercaptopurine; methotrexate; yet another embodiment, treatment with the at least one platinum and platinum coordination complexes such as active agent and treatment with the IDO inhibitor of the cisplatin and carboplatin: vinblastine: etoposide (VP-16); present invention are reduced or discontinued (e.g., lower ifosfamide; mitomycin C; mitoxantrone; Vincristine; Vinore dose, less frequent dosing or shorter treatment regimen). 40 1bine; navelbine; novantrone; teniposide; daunomycin; Oncology-Related Disorders. aminopterin: Xeloda; ibandronate; CPT11: topoisomerase The present invention provides methods for treating and/ inhibitors; difluoromethylornithine (DMFO); retinoic acid; or preventing a proliferative condition, cancer, tumor, or esperamicins; capecitabine; and pharmaceutically accept precancerous disease, disorder or condition with an IDO able salts, acids or derivatives of any of the above. inhibitor and at least one additional therapeutic agent, Such 45 Chemotherapeutic agents also include anti-hormonal as radiation, an immunomodulatory agent or chemothera agents that act to regulate or inhibit hormonal action on peutic agent, or diagnostic agent. Suitable immunomodula tumors such as anti-estrogens, including for example tamox tory agents that may be used in the present invention include ifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hy CD40L, B7, and B7RP1; activating monoclonal antibodies droxytamoxifen, trioxifene, keoxifene, onapristone, and (mAbs) to stimulatory receptors, such as, ant-CD40, anti 50 toremifene; and antiandrogens such as flutamide, niluta CD38, anti-ICOS, and 4-IBB ligand; dendritic cell antigen mide, bicalutamide, leuprolide, and goserelin; and pharma loading (in vitro or in vivo); anti-cancer vaccines such as ceutically acceptable salts, acids or derivatives of any of the dendritic cell cancer vaccines; cytokines/chemokines. Such above. In certain embodiments, combination therapy com as, IL1, IL2, IL12, IL18, ELC/CCL19, SLC/CCL21, MCP prises administration of a hormone or related hormonal 1, IL-4, IL-18, TNF, IL-15, MDC, IFNa/b, M-CSF, IL-3, 55 agent. GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccha Chemotherapeutic agents also include signal transduction rides (LPS); and immune-stimulatory oligonucleotides. inhibitors (STI). The term “signal transduction inhibitor Examples of chemotherapeutic agents include, but are not refers to an agent that selectively inhibits one or more steps limited to, alkylating agents such as thiotepa and cyclos in a signaling pathway. Signal transduction inhibitors (STIs) phosphamide; alkyl Sulfonates Such as buSulfan, improSul 60 of the present invention include: (i) bcr/ablkinase inhibitors fan and piposulfan; aziridines such as benzodopa, carbo (e.g., GLEEVEC); (ii) epidermal growth factor (EGF) quone, meturedopa, and uredopa; ethylenimines and receptor inhibitors, including kinase inhibitors and antibod methylamelamines including altretamine, triethylen ies; (iii) her-2/neu receptor inhibitors (e.g., HERCEPTIN); emelamine, trietylenephosphoramide, triethylenethiophos (iv) inhibitors of Akt family kinases or the Akt pathway phaoramide and trimethylolomelamime; nitrogen mustards 65 (e.g., rapamycin); (V) cell cycle kinase inhibitors (e.g., Such as chiorambucil, chlornaphazine, cholophosphamide, flavopiridol); and (vi) phosphatidyl inositol kinase inhibi estramustine, ifosfamide, mechlorethamine, mechlore tOrS. US 9,598.422 B2 39 40 Additional treatment modalities that may be used in Additional examples of active agents that may be used in combination with an IDO inhibitor include a cytokine or combinations for treating, for example, rheumatoid arthritis, cytokine antagonist, Such as IL-12, IFN, or anti-epidermal include cytokine Suppressive anti-inflammatory drug(s) growth factor receptor, radiotherapy, a monoclonal antibody (CSAIDs); antibodies to, or antagonists of other human against another tumor antigen, a complex of a monoclonal cytokines or growth factors, for example, TNF, LT, IL-1B. antibody and toxin, a T-cell adjuvant, bone marrow trans IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM plant, or antigen presenting cells (e.g., dendritic cell CSF, FGF, or PDGF. therapy). Vaccines (e.g., as a soluble protein or as a nucleic Particular combinations of active agents may interfere at acid encoding the protein) are also provided herein. different points in the autoimmune and Subsequent inflam Cardiovascular Diseases. 10 matory cascade, and include TNF antagonists such as chi meric, humanized or human TNF antibodies, REMICADE, The present invention provides methods for treating and/ anti-TNF antibody fragments (e.g., CDP870), and soluble or preventing certain cardiovascular- and/or metabolic-re p55 or p75 TNF receptors, derivatives thereof, lated diseases, disorders and conditions, as well as disorders p75TNFRIgG (ENBREL) or p55TNFR1 gG (LENER associated therewith, with an IDO inhibitor and at least one 15 CEPT), soluble IL-13 receptor (sIL-13), and also TNFC.- additional therapeutic or diagnostic agent. converting enzyme (TACE) inhibitors; similarly, IL-1 Examples of therapeutic agents useful in combination inhibitors (e.g., Interleukin-1-converting enzyme inhibitors) therapy for the treatment of hypercholesterolemia (and ath may be effective. Other combinations include Interleukin 11, erosclerosis as well) include statins (e.g., CRESTOR, anti-P7s and p-selectin glycoprotein ligand (PSGL). Other LESCOL, LIPITOR, MEVACOR, PRAVACOL, and examples of agents useful in combination with the IDO ZOCOR), which inhibit the enzymatic synthesis of choles inhibitors described herein include interferon-Bla terol; bile acid resins (e.g., COLESTID, LO-CHOLEST, (AVONEX); interferon-?31b (BETASERON); copaxone: PREVALITE, QUESTRAN, and WELCHOL), which hyperbaric oxygen; intravenous immunoglobulin; clabrib sequester cholesterol and prevent its absorption; eZetimibe ine; and antibodies to, or antagonists of other human (ZETIA), which blocks cholesterol absorption; fibric acid 25 cytokines or growth factors (e.g., antibodies to CD40 ligand (e.g., TRICOR), which reduces triglycerides and may mod and CD80). estly increase HDL: niacin (e.g., NIACOR), which modestly Immune Checkpoint Inhibitors. lowers LDL cholesterol and triglycerides; and/or a combi The present invention contemplates the use of the inhibi nation of the aforementioned (e.g., VYTORIN (ezetimibe tors of IDO function described herein in combination with with simvastatin). Alternative cholesterol treatments that 30 additional immune checkpoint inhibitors. may be candidates for use in combination with the IDO The tremendous number of genetic and epigenetic altera inhibitors described herein include various supplements and tions that are characteristic of all cancers provides a diverse herbs (e.g., garlic, policosanol, and guggul). The present set of antigens that the immune system can use to distinguish invention encompasses pharmaceutically acceptable salts, tumor cells from their normal counterparts. In the case of T acids or derivatives of any of the above. 35 cells, the ultimate amplitude (e.g., levels of cytokine pro Immune- and Inflammatory-Related Disorders. duction or proliferation) and quality (e.g., the type of The present invention provides methods for treating and/ immune response generated. Such as the pattern of cytokine or preventing immune- and/or inflammatory-related dis production) of the response, which is initiated through eases, disorders and conditions, as well as disorders asso antigen recognition by the T-cell receptor (TCR), is regu ciated therewith, with an IDO inhibitor and at least one 40 lated by a balance between co-stimulatory and inhibitory additional therapeutic or diagnostic agent. signals (immune checkpoints). Under normal physiological Examples of therapeutic agents useful in combination conditions, immune checkpoints are crucial for the preven therapy include, but are not limited to, the following: tion of autoimmunity (i.e., the maintenance of self-toler non-steroidal anti-inflammatory drug (NSAID) such as aspi ance) and also for the protection of tissues from damage rin, ibuprofen, and other propionic acid derivatives (almi 45 when the immune system is responding to pathogenic infec noprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, tion. The expression of immune checkpoint proteins can be fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, dysregulated by tumors as an important immune resistance miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, mechanism. Suprofen, tiaprofenic acid, and tioxaprofen), acetic acid T cells have been the major focus of efforts to therapeu derivatives (indomethacin, acemetacin, alclofenac, clidanac, 50 tically manipulate endogenous antitumor immunity because diclofenac, fenclofenac, fenclozic acid, fentiazac, fuiro of i) their capacity for the selective recognition of peptides fenac, ibufenac, isoxepac, oXpinac, Sulindac, tiopinac, tol derived from proteins in all cellular compartments; ii) their metin, Zidometacin, and Zomepirac), fenamic acid deriva capacity to directly recognize and kill antigen-expressing tives (flufenamic acid, meclofenamic acid, mefenamic acid, cells (by CD8+ effector T cells; also known as cytotoxic T niflumic acid and tolfenamic acid), biphenylcarboxylic acid 55 lymphocytes (CTLs)); and iii) their ability to orchestrate derivatives (diflunisal and flufenisal), oxicams (isoxicam, diverse immune responses by CD4+ helper T cells, which piroXicam, Sudoxicam and tenoxican), salicylates (acetyl integrate adaptive and innate effector mechanisms. In the salicylic acid, Sulfasalazine) and the pyrazolones (apaZone, clinical setting, the blockade of immune checkpoints— bezpiperylon, feprazone, mofebutaZone, oxyphenbutaZone, which results in the amplification of antigen-specific T cell phenylbutaZone). Other combinations include cyclooxy 60 responses—has shown to be a promising approach in human genase-2 (COX-2) inhibitors. cancer therapeutics. Other active agents for combination include steroids such T cell-mediated immunity includes multiple sequential as prednisolone, prednisone, methylprednisolone, betame steps, each of which is regulated by counterbalancing stimu thasone, dexamethasone, or hydrocortisone. Such a combi latory and inhibitory signals in order to optimize the nation may be especially advantageous since one or more 65 response. While nearly all inhibitory signals in the immune adverse effects of the steroid can be reduced or even response ultimately modulate intracellular signaling path eliminated by tapering the steroid dose required. ways, many are initiated through membrane receptors, the US 9,598.422 B2 41 42 ligands of which are either membrane-bound or soluble tant family of membrane-bound ligands that bind to (cytokines). While co-stimulatory and inhibitory receptors co-stimulatory or co-inhibitory receptors is the B7 family, and ligands that regulate T-cell activation are frequently not which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD over-expressed in cancers relative to normal tissues, inhibi L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and tory ligands and receptors that regulate T cell effector 5 B7-H6. Another family of membrane bound ligands that functions in tissues are commonly overexpressed on tumor bind to co-stimulatory or co-inhibitory receptors is the TNF cells or on non-transformed cells associated with the tumor family of molecules that bind to cognate TNF receptor microenvironment. The functions of the soluble and mem family members, which includes CD40 and CD40L, OX-40, brane-bound receptor-ligand immune checkpoints can be OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 modulated using agonistantibodies (for co-stimulatory path- 10 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, ways) or antagonist antibodies (for inhibitory pathways). TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/ Thus, in contrast to most antibodies currently approved for Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, cancer therapy, antibodies that block immune checkpoints BCMA, LTBR, LIGHT, DcR3, HVEM, VEGI/TL1A, do not target tumor cells directly, but rather target lympho TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, cyte receptors or their ligands in order to enhance endog.- 15 Lymphotoxin C/TNFB, TNFR2, TNFC., LTBR, Lympho enous antitumor activity. See Pardoll, (April 2012) Nature toxin c. 1 B2, FAS, FASL, RELT, DR6, TROY, NGFR. Rev. Cancer 12:252-64. In another aspect, the immuno-oncology agent is a Examples of immune checkpoints (ligands and receptors), cytokine that inhibits T cell activation (e.g., IL-6, IL-10, Some of which are selectively upregulated in various types TGF-B, VEGF, and other immunosuppressive cytokines) or of tumor cells, that are candidates for blockade include PD1 20 a cytokine that stimulates T cell activation, for stimulating (programmed cell death protein 1); PDL1 (PD1 ligand); an immune response. BTLA (B and T lymphocyte attenuator); CTLA4 (cytotoxic In one aspect, T cell responses can be stimulated by a T-lymphocyte associated antigen 4); TIM3 (T-cell mem combination of the claimed IDO inhibitors and one or more brane protein 3); LAG3 (lymphocyte activation gene 3): of (i) an antagonist of a protein that inhibits T cell activation A2aR (adenosine A2a receptor A2aR); and Killer Inhibitory 25 (e.g., immune checkpoint inhibitors) such as CTLA-4, PD-1, Receptors, which can be divided into two classes based on PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, their structural features: i) killer cell immunoglobulin-like BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, receptors (KIRs), and ii) C-type lectin receptors (members 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4 of the type II transmembrane receptor family). Other less and/or (ii) an agonist of a protein that stimulates T cell well-defined immune checkpoints have been described in 30 activation such as B7-1, B7-2, CD28, 4-1BB (CD137), the literature, including both receptors (e.g., the 2B4 (also 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, known as CD244) receptor) and ligands (e.g., certain B7 CD70, CD27, CD40, DR3 and CD2. Other agents that can family inhibitory ligands such B7-H3 (also known as be combined with the IDO inhibitors of the present inven CD276) and B7-H4 (also known as B7-S1, B7x and tion for the treatment of cancer include antagonists of VCTN1)). See PardoII, (April 2012) Nature Rev. Cancer 35 inhibitory receptors on NK cells or agonists of activating 12:252-64. receptors on NK cells. For example, compounds herein can The present invention contemplates the use of the inhibi be combined with antagonists of KIR, such as lirilumab. tors of IDO function described herein in combination with Yet other agents for combination therapies include agents inhibitors of the aforementioned immune-checkpoint recep that inhibit or deplete macrophages or monocytes, including tors and ligands, as well as yet-to-be-described immune- 40 but not limited to CSF-1R antagonists such as CSF-1R checkpoint receptors and ligands. Certain modulators of antagonist antibodies including RG7155 (WO11/70024, immune checkpoints are currently available, whereas others WO11/107553, WO11/131407, WO 13/87699, WO13/ are in late-stage development. To illustrate, when it was 119716, WO 13/132044) or FPA-008 (WO11/140249; approved for the treatment of melanoma in 2011, the fully WO13169264; WO14/036357). humanized CTLA4 monoclonal antibody ipilimumab (YER- 45 In another aspect, the claimed IDO inhibitors can be used VOY; Bristol-Myers Squibb) became the first immune with one or more of agonistic agents that ligate positive checkpoint inhibitor to receive regulatory approval in the costimulatory receptors, blocking agents that attenuate sig US. Fusion proteins comprising CTLA4 and an antibody naling through inhibitory receptors, antagonists, and one or (CTLA4-Ig: abatcept (ORENCIA: Bristol-Myers Squibb)) more agents that increase systemically the frequency of have been used for the treatment of rheumatoid arthritis, and 50 anti-tumor T cells, agents that overcome distinct immune other fusion proteins have been shown to be effective in Suppressive pathways within the tumor microenvironment renal transplantation patients that are sensitized to Epstein (e.g., block inhibitory receptor engagement (e.g., PD-L1/ Barr Virus. PD1 antibodies are also available for the treat PD-1 interactions), deplete or inhibit Tregs (e.g., using an ment of cancer, including for example nivolumab (Bristol anti-CD25 monoclonal antibody (e.g., daclizumab) or by ex Myers Squibb) and pembroluzimab (Merck), and anti-PDL1 55 vivo anti-CD25 bead depletion), or reverse/prevent T cell antibodies are also being evaluated (e.g., MPDL3280A anergy or exhaustion) and agents that trigger innate immune (Roche)). Nivolumab (Opdivo(R) has shown promise in activation and/or inflammation at tumor sites. patients with melanoma, lung and kidney cancer, as well as In one aspect, the immuno-oncology agent is a CTLA-4 multiple other malignancies. antagonist, such as an antagonistic CTLA-4 antibody. Suit In one aspect of the present invention, the claimed IDO 60 able CTLA-4 antibodies include, for example, YERVOY inhibitors are combined with an immuno-oncology agent (ipilimumab) or tremelimumab. that is (i) an agonist of a stimulatory (including a co In another aspect, the immuno-oncology agent is a PD-1 stimulatory) receptor or (ii) an antagonist of an inhibitory antagonist, such as an antagonistic PD-1 antibody. Suitable (including a co-inhibitory) signal on T cells, both of which PD-1 antibodies include, for example, OPDIVO result in amplifying antigen-specific T cell responses. Cer- 65 (nivolumab), KEYTRUDA (pembrolizumab/lambroli tain of the stimulatory and inhibitory molecules are mem Zumab), or MEDI-0680 (AMP-514; WO2012/145493). The bers of the immunoglobulin super family (IgSF). One impor immuno-oncology agent may also include pidilizumab (CT US 9,598.422 B2 43 44 011), though its specificity for PD-1 binding has been Virenz, nevirapine); nucleotide analogue reverse tran questioned. Another approach to target the PD-1 receptor is Scriptase inhibitors; and agents that prevent release of viral the recombinant protein composed of the extracellular particles (e.g., Zanamivir and oseltamivir). Treatment and/or domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1. prevention of certain viral infections (e.g., HIV) frequently called AMP-224 entail a group ("cocktail”) of antiviral agents. In another aspect, the immuno-oncology agent is a PD-L1 Other antiviral agents contemplated for use in combina antagonist, such as an antagonistic PD-L1 antibody. Suitable tion with an IDO inhibitor include, but are not limited to, the PD-L1 antibodies include, for example, MPDL3280A following: abacavir, adefovir, amantadine, amprenavir, amp (RG7446; WO2010/077634), durvalumab (MEDI4736), ligen, arbidol, atazanavir, atripla, boceprevirertet, cidofovir, BMS-936559 (WO2007/005874), and MSB0010718C 10 combivir, darunavir, delavirdine, didanosine, docosanol, (WO2013/79174). edoxudine, emitricitabine, enfuvirtide, entecavir, famciclo In another aspect, the immuno-oncology agent is a LAG-3 Vir, fosamprenavir, foScarnet, fosfonet, ganciclovir, ibacit antagonist, Such as an antagonistic LAG-3 antibody. Suit abine, imunovir, idoxuridine, imiquimod, indinavir, inosine, able LAG3 antibodies include, for example, BMS-98.6016 various interferons (e.g., peginterferon alfa-2a), lopinavir, (WO10/19570, WO14/08218), or IMP-731 or IMP-321 15 loviride, maraviroc, moroxydine, methisaZone, nelfinavir, (WO08/132601, WO09/44273). nexavir, penciclovir, peramivir, pleconaril, podophyllotoxin, In another aspect, the immuno-oncology agent is a CD137 raltegravir, ribavirin, ritonavir, pyramidine, saquinavir, sta (4-IBB) agonist, such as an agonistic CD137 antibody. Vudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, Suitable CD137 antibodies include, for example, urelumab tromantadine, truvada, Valaciclovir, Valganciclovir, Vicrivi and PF-05082566 (WO12/32433). roc, Vidarabine, Viramidine, and Zalcitabine. In another aspect, the immuno-oncology agent is a GITR The present invention encompasses pharmaceutically agonist, such as an agonistic GITR antibody. Suitable GITR acceptable salts, acids or derivatives of any of the above. antibodies include, for example, BMS-986153, BMS Parasitic Disorders. 986156, TRX-518 (WO06/105021, WO09/009116) and The present invention contemplates the use of the inhibi MK-4166 (WO11/028683). 25 tors of IDO function described herein in combination with In another aspect, the immuno-oncology agent is an antiparasitic agents. Such agents include, but are not limited OX40 agonist, such as an agonistic OX40 antibody. Suitable to, thiabendazole, pyrantel pamoate, mebendazole, praZiqu OX40 antibodies include, for example, MEDI-6383 or antel, niclosamide, bithionol, oxamniquine, metrifonate, MEDI-6469. ivermectin, albendazole, eflornithine, melarsoprol, pentami In another aspect, the immuno-oncology agent is an 30 dine, benznidazole, nifurtimoX, and nitroimidazole. The OX40L antagonist, such as an antagonistic OX40 antibody. skilled artisan is aware of other agents that may find utility Suitable OX40L antagonists include, for example, RG-7888 for the treatment of parasitic disorders. (WO06/029879). The present invention encompasses pharmaceutically In another aspect, the immuno-oncology agent is a CD40 acceptable salts, acids or derivatives of any of the above. agonist, such as an agonistic CD40 antibody. In yet another 35 Bacterial Infections. embodiment, the immuno-oncology agent is a CD40 antago Embodiments of the present invention contemplate the nist, such as an antagonistic CD40 antibody. Suitable CD40 use of the IDO inhibitors described herein in combination antibodies include, for example, lucatumumab or dacetu with agents useful in the treatment or prevention of bacterial Zumab. disorders. Antibacterial agents can be classified in various In another aspect, the immuno-oncology agent is a CD27 40 manners, including based on mechanism of action, based on agonist, such as an agonistic CD27 antibody. Suitable CD27 chemical structure, and based on spectrum of activity. antibodies include, for example, Varlilumab. Examples of antibacterial agents include those that target the In another aspect, the immuno-oncology agent is bacterial cell wall (e.g., cephalosporins and penicillins) or MGA271 (to B7H3) (WO11/109400). the cell membrane (e.g., polymyxins), or interfere with The present invention encompasses pharmaceutically 45 essential bacterial enzymes (e.g., Sulfonamides, rifamycins, acceptable salts, acids or derivatives of any of the above. and quinolines). Most antibacterial agents that target protein Viral Diseases. synthesis (e.g., tetracyclines and macrollides) are bacterio The present invention provides methods for treating and/ static, whereas agents such as the aminoglycoside are bac or preventing viral diseases, disorders and conditions, as tericidal. Another means of categorizing antibacterial agents well as disorders associated therewith, with an IDO inhibitor 50 is based on their target specificity: “narrow-spectrum’ and at least one additional therapeutic or diagnostic agent agents target specific types of bacteria (e.g., Gram-positive (e.g., one or more other antiviral agents and/or one or more bacteria such as Streptococcus), while “broad-spectrum’ agents not associated with viral therapy). agents have activity against a broader range of bacteria. The Such combination therapy includes anti-viral agents tar skilled artisan is aware of types of anti-bacterial agents that geting various viral life-cycle stages and having different 55 are appropriate for use in specific bacterial infections. mechanisms of action, including, but not limiting to, the The present invention encompasses pharmaceutically following: inhibitors of viral uncoating (e.g., amantadine acceptable salts, acids or derivatives of the agents (and and rimantidine); reverse transcriptase inhibitors (e.g., acy members of the classes of agents) set forth above. clovir, Zidovudine, and lamivudine); agents that target inte Dosing grase; agents that block attachment of transcription factors 60 The IDO inhibitors of the present invention may be to viral DNA; agents (e.g., antisense molecules) that impact administered to a subject in an amount that is dependent translation (e.g., fomivirsen); agents that modulate transla upon, for example, the goal of administration (e.g., the tion/ribozyme function; protease inhibitors; viral assembly degree of resolution desired); the age, weight, sex, and modulators (e.g., rifampicin); antiretrovirals such as, for health and physical condition of the subject to which the example, nucleoside analogue reverse transcriptase inhibi 65 formulation is being administered; the route of administra tors (e.g., azidothymidine (AZT), ddl, ddC, 3TC. d4T): tion; and the nature of the disease, disorder, condition or non-nucleoside reverse transcriptase inhibitors (e.g., efa symptom thereof. The dosing regimen may also take into US 9,598.422 B2 45 46 consideration the existence, nature, and extent of any can be provided in a form that is ready for use (e.g., a tablet adverse effects associated with the agent(s) being adminis or capsule) or in a form requiring, for example, reconstitu tered. Effective dosage amounts and dosage regimens can tion or dilution (e.g., a powder) prior to administration. readily be determined from, for example, safety and dose When the IDO inhibitors are in a form that needs to be escalation trials, in Vivo studies (e.g., animal models), and reconstituted or diluted by a user, the kit may also include other methods known to the skilled artisan. diluents (e.g., Sterile water), buffers, pharmaceutically In general, dosing parameters dictate that the dosage acceptable excipients, and the like, packaged with or sepa amount be less than an amount that could be irreversibly rately from the IDO inhibitors. When combination therapy is toxic to the subject (the maximum tolerated dose (MTD)) contemplated, the kit may contain the several agents sepa and not less than an amount required to produce a measur 10 rately or they may already be combined in the kit. Each able effect on the subject. Such amounts are determined by, component of the kit may be enclosed within an individual for example, the pharmacokinetic and pharmacodynamic container, and all of the various containers may be within a parameters associated with ADME, taking into consider single package. A kit of the present invention may be ation the route of administration and other factors. designed for conditions necessary to properly maintain the An effective dose (ED) is the dose or amount of an agent 15 components housed therein (e.g., refrigeration or freezing). that produces a therapeutic response or desired effect in A kit may contain a label or packaging insert including some fraction of the subjects taking it. The “median effective identifying information for the components therein and dose” or ED50 of an agent is the dose or amount of an agent instructions for their use (e.g., dosing parameters, clinical that produces a therapeutic response or desired effect in 50% pharmacology of the active ingredient(s), including mecha of the population to which it is administered. Although the nism of action, pharmacokinetics and pharmacodynamics, ED50 is commonly used as a measure of reasonable expect adverse effects, contraindications, etc.). Labels or inserts can ance of an agent’s effect, it is not necessarily the dose that include manufacturer information Such as lot numbers and a clinician might deem appropriate taking into consideration expiration dates. The label or packaging insert may be, e.g., all relevant factors. Thus, in some situations the effective integrated into the physical structure housing the compo amount is more than the calculated ED50, in other situations 25 nents, contained separately within the physical structure, or the effective amount is less than the calculated ED50, and in affixed to a component of the kit (e.g., an ampule, tube or still other situations the effective amount is the same as the vial). calculated ED50. Labels or inserts can additionally include, or be incorpo In addition, an effective dose of the IDO inhibitors of the rated into, a computer readable medium, Such as a disk (e.g., present invention may be an amount that, when administered 30 hard disk, card, memory disk), optical disk Such as CD- or in one or more doses to a subject, produces a desired result DVD-ROM/RAM, DVD, MP3, magnetic tape, or an elec relative to a healthy subject. For example, for a subject trical storage media such as RAM and ROM or hybrids of experiencing a particular disorder, an effective dose may be these such as magnetic/optical storage media, FLASH media one that improves a diagnostic parameter, measure, marker or memory-type cards. In some embodiments, the actual and the like of that disorder by at least about 5%, at least 35 instructions are not present in the kit, but means for obtain about 10%, at least about 20%, at least about 25%, at least ing the instructions from a remote source, e.g., via the about 30%, at least about 40%, at least about 50%, at least internet, are provided. about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, where 100% is defined as the Experimental diagnostic parameter, measure, marker and the like exhib 40 ited by a normal Subject. The following Examples are put forth so as to provide For administration of an oral agent, the compositions can those of ordinary skill in the art with a complete disclosure be provided in the form of tablets, capsules and the like and description of how to make and use the present inven containing from 1.0 to 1000 milligrams of the active ingre tion, and are not intended to limit the scope of what the dient, particularly 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 45 inventors regard as their invention, nor are they intended to 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, represent that the experiments below were performed or that 750.0, 800.0, 900.0, and 1000.0 milligrams of the active they are all of the experiments that may be performed. It is ingredient. to be understood that exemplary descriptions written in the In certain embodiments, the dosage of the desired IDO present tense were not necessarily performed, but rather that inhibitor is contained in a “unit dosage form'. The phrase 50 the descriptions can be performed to generate data and the “unit dosage form” refers to physically discrete units, each like of a nature described therein. Efforts have been made to unit containing a predetermined amount of the IDO inhibi ensure accuracy with respect to numbers used (e.g., tor, either alone or in combination with one or more addi amounts, temperature, etc.), but some experimental errors tional agents, sufficient to produce the desired effect. It will and deviations should be accounted for. be appreciated that the parameters of a unit dosage form will 55 Unless indicated otherwise, parts are parts by weight, depend on the particular agent and the effect to be achieved. molecular weight is weight average molecular weight, tem Kits perature is in degrees Celsius (° C.), and pressure is at or The present invention also contemplates kits comprising near atmospheric. Standard abbreviations are used, includ an IDO inhibitor, and pharmaceutical compositions thereof. ing the following: wt wildtype; bp=base pair(s): The kits are generally in the form of a physical structure 60 kb-kilobase(s); nt nucleotides(s): aa amino acid(s); S or housing various components, as described below, and may sec-second(s); min minute(s); h or hr hour(s): be utilized, for example, in practicing the methods described ng nanogram; Lig microgram, mg-milligram; g. gram; above. kg kilogram; dl or dL deciliter; ul or uL-microliter; ml or A kit can include one or more of the IDO inhibitors mL milliliter; 1 O L=liter; uM-micromolar; disclosed herein (provided in, e.g., a sterile container), 65 mM=millimolar; M=molar; kDa-kilodalton; which may be in the form of a pharmaceutical composition i.m. intramuscular(ly); i.p. intraperitoneal (ly); SC or suitable for administration to a subject. The IDO inhibitors SQ-Subcutaneous(ly); QD-daily; BID=twice daily; US 9,598.422 B2 47 48 QW-weekly; QM-monthly: HPLC-high performance liq basis of a cell-based assay, and may be quantified via uid chromatography: BW-body weight; U-unit; ins—not LCMS/MS as an alternative to UV/Vis detection. statistically significant; PBS-phosphate-buffered saline: Western Blot Analyses. IHC-immunohistochemistry; DMEM-Dulbecco's Modifi Groups of 1,000-1,200 islets incubated for 24h in Miami cation of Eagle's Medium; medium in the presence of cytokines can be harvested and EDTA ethylenediaminetetraacetic acid. Sonicated in PBS as above, and 50 ug protein samples can Materials and Methods be electrophoresed on 10% SDS-PAGE gels. COS7 cells The following general materials and methods were used, (0.6x10° cells/60 mm3 petri dish) transfected with human where indicated, or may be used in the Examples below: IDO plasmid (3 Jug) or empty vector cells can be used as Standard methods in molecular biology are described in 10 positive and negative controls, respectively. Proteins can be the scientific literature (see, e.g., Sambrook et al., Molecular Cloning. Third Edition, Cold Spring Harbor Laboratory transferred electrophoretically onto polyvinylidine fluoride Press, Cold Spring Harbor, N.Y. (2001); and Ausubel et al., membranes by semidry method and blocked for 1 h with 5% Current Protocols in Molecular Biology, Vols. 1-4, John (w/v) nonfat dry milk in Tris-buffered saline and 0.1% Wiley and Sons, Inc. New York, N.Y. (2001), which 15 Tween and then incubated overnight with anti-human mouse describes cloning in bacterial cells and DNA mutagenesis IDO antibody (1:500; Chemicon, Temecula, Calif.), phos (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), pho-STAT. p91, and STAT. p91 (1:500, Zymed, San glycoconjugates and protein expression (Vol. 3), and bioin Francisco, Calif.). Immunoreactive proteins can be visual formatics (Vol. 4)). ized with ECL PLUS(R) Western blotting detection reagent The scientific literature describes methods for protein (Amersham BioSciences, Buckinghamshire, U.K.) after purification, including immunoprecipitation, chromatogra incubation for 1 h with anti-mouse horseradish peroxidase phy, electrophoresis, centrifugation, and crystallization, as conjugated secondary antibody (Jackson Immunolabs, West well as chemical analysis, chemical modification, post Grove, Pa.). translational modification, production of fusion proteins, Immunohistochemical Detection of IDO. and glycosylation of proteins (see, e.g., Coligan et al., 25 Islets can be fixed in 4% paraformaldehyde in PBS Current Protocols in Protein Science, Vols. 1-2, John Wiley (Invitrogen) for 1 h, immobilized in molten 10% porcine and Sons, Inc., NY (2000)). skin gelatin blocks (37° C.), and embedded in optimal Software packages and databases for determining, e.g., cutting temperature compound. Immunofluorescent staining antigenic fragments, leader sequences, protein folding, func on islet tissue can be performed on 7 um sections that were tional domains, glycosylation sites, and sequence align 30 stained with antibodies raised against pancreatic duodenal ments, are available (see, e.g., GCG(R) Wisconsin Package homeobox 1 (PDX1) and IDO. Antigen retrieval can be (Accelrys, Inc., San Diego, Calif.); and DECYPHER(R) performed in a water bath for 30 min in a buffer containing (TimeLogic Corp., Crystal Bay, Nev.). 10 mmol/1 Tris and 1 mmol/l EDTA (pH 9.0) at 97°C. The The literature is replete with assays and other experimen sections can be blocked for 1 h with 5% normal goat serum tal techniques that can serve as a basis for evaluation of the 35 in PBS. The tissues can then be reacted with mouse mono compounds described herein. clonal anti-human IDO antibody (1:20; Chemicon) and goat An IDO enzyme assay and cellular production of kynure polyclonal anti-human PDX1 antibody (1:2,000; which may nine (KYN) is described in Sarkar, S. A. et al., Diabetes, be requested from Dr. Chris Wright, School of Medicine, 56:72-79 (2007). Briefly, all chemicals can be purchased Vanderbilt, Tenn.) overnight at room temperature in a humid from Sigma-Aldrich (St. Louis, Mo.) unless specified oth 40 chamber. Secondary antibodies anti-goat (labeled with Cy3) erwise. Groups of 1,000 human islets can be cultured for 24 and anti-mouse (labeled with Cy2) can be purchased from h in 1 mL medium with cytokines, recovered by centrifu Jackson Immunolabs and can be used at a concentration of gation for 5 min at 800xg and sonicated in 150 uL PBS 1:200. The nuclei can be stained with Hoechst 33258 containing a protease inhibitor cocktail (Set 2: Calbiochem, (Molecular Probes, Eugene, Oreg.). Images can be acquired EMD Biosciences, San Diego, Calif.). The sonicate can be 45 by Intelligent Imaging System software from an Olympus centrifuged for 10 min at 10,000xg, and the supernatant can 1X81 inverted motorized microscope equipped with Olym be assayed in triplicate by incubating a 40 ul sample with an pus DSU (spinning disk confocal) and Hamamatsu ORCA equal volume of 100 mmol/L potassium phosphate buffer, IIER monochromatic CCD camera. pH 6.5, containing 40 mmol/L ascorbic acid (neutralized to Alternative means for evaluating the IDO inhibitors of the pH 7.0), 100 umol/L methylene blue, 200 g/mL catalase, 50 present invention are described in WO 2010/0233166 and and 400 umol/l L-Trp for 30 min at 37°C. The assay can be are summarized hereafter. terminated by the addition of 16 LL 30% (w/v) trichloro Biochemical Assay. acetic acid (TCA) and further incubated at 60° C. for 15 min cDNA clones for both human and mouse IDO have been to hydrolyze N-formylkynurenine to KYN. The mixture can isolated and Verified by sequencing and are commercially then be centrifuged at 12,000 rpm for 15 min, and KYN can 55 available. In order to prepare IDO for biochemical studies, be quantified by mixing equal Volume of Supernatant with C-terminal His-tagged IDO protein can be produced in E. 2% (w/v) Ehrlich's reagent in glacial acetic acid in 96-well coli using the IPTG-inducible plT5a vector system and microtiter plate and reading the absorbance at 480 nm using isolated over a nickel column. The yield of the partially L-KYN as standard. Protein in the islet samples can be purified protein can be verified by gel electrophoresis and quantified by Bio-Rad Protein assay at 595 nm. For the 60 the concentration estimated by comparison to protein stan detection of L-KYN in the islet culture supernatants, pro dards. To assay IDO enzymatic activity, a 96-well plate teins can be precipitated with 5% (w/v) TCA and centrifuged spectrophotometric assay for kynurenine production can be at 12,000 rpm for 15 min, and determination of KYN in the run following published procedures (see, e.g., Littlejohn, T. Supernatant with Ehrlich's reagent can be determined as K. et al., Prot. Exp. Purif., 19:22-29 (2000)). To screen for described above. IL-4 (10 ug/mL: 500-2,000 units/mL) and 65 IDO inhibitory activity, compounds can be evaluated at a 1-C.-methyl Trp (1-MT; 40 umol/L) can be added to the single concentration of for example, 200 LM against 50 ng incubation media as indicated. This assay can also form the of IDO enzyme in 100 uL reaction volumes with tryptophan US 9,598.422 B2 49 50 added at increasing concentrations at, for example, 0, 2, 20, rate: 0.8 ml/min: Mobile Phase A: 100% Water, 0.05% TFA: and 200 uM. Kynurenine production can be measured at 1 Mobile Phase B: 100% Acetonitrile, 0.05% TFA. hour. Method B: Column: Waters Acquity UPLC BEH C18, Cell-Based Assay. 2.1 x50mm, 1.7-lum particles; Mobile Phase A: 5:95 acetoni COS-1 cells can be transiently transfected with a CMV trile: water with 10 mMammonium acetate; Mobile Phase B: promoter-driven plasmid expressing IDO cDNA using Lipo 95:5 acetonitrile:water with 10 mM ammonium acetate; fectamine 2000 (Invitrogen) as recommended by the manu Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, facturer. A companion set of cells can be transiently trans then a 0.75-minute hold at 100% B; Flow: 1.00 mL/min: fected with TDO-expressing plasmid. Forty-eight hours Detection: UV at 220 nm. post-transfection, the cells can be apportioned into a 96-well 10 Method C: Waters SFC-100 MS, Column: Chiral OJ-H format at 6x10" cells per well. The following day, the wells 25x3 cm ID, 5um Flow rate: 100.0 mL/min, Mobile Phase: can be washed and new media (phenol red free) containing 80/20 CO/MeOH, Detector Wavelength: 220 nm. 20 ug/mL tryptophan can be added together with inhibitor. Method D: Aurora analytical SFC, Column: Chiral OJ-H The reaction can be stopped at 5 hours and the Supernatant 15 250x4.6 mm ID, 5 um, Flow rate: 2.0 mL/min, Mobile removed and spectrophotometrically-assayed for kynure Phase: 80/20 CO/MeOH. nine as previously described for the enzyme assay. To obtain Method E: Berger Prep SFC, Column: Chiral AS 25x3 cm initial confirmation of IDO activity, compounds can be ID, 5 um Flow rate: 85.0 mL/min, Mobile Phase: 82/18 evaluated at a single concentration of for example, 100 LM. CO/MeOH w/ 0.1% DEA, Detector Wavelength: 220 nm. More extensive dose-escalation profiles can be collected for Method F: Aurora analytical SFC, Column: Chiral AS select compounds. 250x4.6 mm ID, 5 um, Flow rate: 2.0 mL/min, Mobile Pharmacodynamic and Pharmacokinetic Evaluation. Phase: 80/20 CO/MeOH w/ 0.1% DEA. A pharmacodynamic assay can be based on measuring Method G: Berger Prep SFC, Column: Chiral AS 25x3 cm serum levels of both kynurenine and tryptophan, and calcu ID, 5 um Flow rate: 85.0 mL/min, Mobile Phase: 86/14 lating the kynurenine/tryptophan ratio provides an estimate 25 CO/MeOH, Detector Wavelength: 220 nm. of IDO activity that is independent of baseline tryptophan Method H: Aurora analytical SFC, Column: Chiral AS levels. Serum tryptophan and kynurenine levels can be 250x4.6 mm ID, 5 um, Flow rate: 2.0 mL/min, Mobile determined by HPLC analysis, and serum compound levels Phase: 85/15 CO/MeOH. can optionally also be determined in the same HPLC run. Method I: Column: Waters Acquity UPLC BEH C18, Compounds can be initially evaluated by challenging 30 2.1 x50mm, 1.7-lum particles; Mobile Phase A: 5:95 acetoni mice with LPS and then subsequently administering a bolus trile: water with 0.1% trifluoroacetic acid; Mobile Phase B: dose of compound at the time that the serum kynurenine 95:5 acetonitrile:water with 0.1% trifluoroacetic acid: Tem level plateaus. As the kynurenine pool is rapidly turned over perature: 50° C.; Gradient: 0-100% B over 3 minutes, then with a half-life in serum of less than 10 minutes, pre-existing a 0.75-minute hold at 100% B; Flow: 1.0 mL/min: Detec kynurenine is not expected to unduly mask the impact that 35 tion: UV at 220 nm. an IDO inhibitor has on kynurenine production. Each Method J: Preparative Chromatographic Conditions: experiment can include non-LPS-exposed mice (to deter Instrument: Berger Prep SFC MGII (LVL-L4021 Lab) Col mine baseline kynurenine levels against which to compare umn: Chiral IC 25x3 cm ID, 5um; Flow rate: 85.0 mL/min: the other mice) and a set of LPS-exposed mice dosed with Mobile Phase: 74/26 CO/MeOH: Detector Wavelength: vehicle alone (to provide a positive control for IDO activa 40 220 nm. tion). Each compound can initially be evaluated in mice at Method K. Preparative Chromatographic Conditions: a single high i.p. bolus dose in the range of at least 100 Instrument: Berger Prep SFC MGII (LVL-L4021 Lab) Col mg/kg. Blood can be collected at defined time intervals (for umn: Chiral IC 25x3 cm ID, 5um; Flow rate: 85.0 mL/min: example, 50 uL sample at 5, 15, 30 min. 1, 2, 4, 6, 8, and Mobile Phase: 75/25 CO/MeOH hold for 18 minutes, 60/40 24hr. following compound administration) for HPLC analy 45 CO/MeOH hold for 11 minutes, 75/25 CO/MeOH hold for sis of kynurenine and tryptophan levels (pharmacodynamic 3 minutes; Detector Wavelength: 220 nm. analysis) as well as for the level of compound (pharmacoki Method L: Preparative Conditions: Berger SFC MGII; netic analysis). From the pharmacokinetic data the peak Stage-1: Column: Chiral OD-H 25x3 cm ID, 5-lum particles: serum concentration of compound achieved can be deter Mobile Phase: 82/18 CO/MeOH: Detector Wavelength: mined as well as the estimated rate of clearance. By com 50 220 mm; Flow: 85 mL/min. Stage-2: Chiral IF 25x3 cm ID, paring the level of compound in serum relative to the 5-lum particles; Mobile Phase: 80/20 CO/MeOH: Detector kynurenine/tryptophan ratio at various time points, the Wavelength: 220 nm, Flow: 85 mL/min. Analytical Condi effective ICs for IDO inhibition in vivo can be roughly tions: Aurora analytical SFC: Stage-1: Column: Chiral estimated. Compounds exhibiting efficacy can be evaluated OD-H 250x4.6 mm ID, 5 um; Mobile Phase: 80/20 CO/ to determine a maximum dose that achieves 100% IDO 55 MeOH: Flow: 2.0 mL/min: Stage-2: Column: Chiral IF inhibition at the peak concentration. 250x4.6 mm ID, 5 um; Mobile Phase: 80/20 CO/MeOH: Flow: 2.0 mL/min. Tr corresponds to the analytical condi HPLC/MS and Preparatory/Analytical HPLC tion. Methods Employed in Characterization or Method M: Preparative Conditions: Berger SFC MGII; Purification of Examples 60 Stage-1: Column: Chiral OD-H 25x3 cm ID, 5-lum particles: Mobile Phase: 80/20 CO/MeOH: Detector Wavelength: Analytical HPLC/MS was performed using the following 220 mm; Flow: 85 mL/min. Stage-2: Chiral IF 25x3 cm ID, methods: 5-um particles; Mobile Phase: 80/20 CO/MeOH: Detector Method A: Waters Acquity SDS using the following Wavelength: 220 nm, Flow: 85 mL/min. Analytical Condi method: Linear Gradient of 2% to 98% Solvent B over 1.7 65 tions: Aurora analytical SFC: Stage-1: Column: Chiral min: UV visualization at 220 nmi; Column: BEH C18 2.1 OD-H 250x4.6 mm ID, 5 um; Mobile Phase: 80/20 CO/ mmx50mm; 1.7 um particle (Heated to Temp. 50° C.); Flow MeOH: Flow: 2.0 mL/min: Stage-2: Column: Chiral IF US 9,598.422 B2 51 52 250x4.6 mm ID, 5 um; Mobile Phase: 80/20 CO/MeOH: SFC: Column: Chiral WHELK-OR 250x4.6 mm ID, 5um: Flow: 2.0 mL/min. Tr corresponds to the analytical condi Mobile Phase: 80/20 CO/MeOH: Flow: 2.0 mL/min: Tr tion. corresponds to the analytical condition. Method N: Preparative Conditions: Berger SFC MGII; Method W: Preparative Conditions: Berger SFC MGH; Column: WHELK-OR 1 KROMASILR 25x3 cm ID, 5-lum 5 Column: Chiral IC 25x3 cm ID, 5-um: Mobile Phase: 85/15 particles: Mobile Phase: 80/20 CO/MeOH: Detector Wave CO/MeOH: Detector Wavelength: 220 mm; Flow: 85 length: 220 nm; Flow: 85 mL/min. Analytical Conditions: mL/min. Analytical Conditions: Aurora analytical SFC: Col Aurora analytical SFC: Column: WHELK-OR 1 KROMA umn: Chiral IC 250x4.6 mm ID, 5um; Mobile Phase: 85/15 SILR 250x4.6 mm ID, 5 um; Mobile Phase: 80/20 CO/ CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the MeOH: Flow: 2.0 mL/min: Tr corresponds to the analytical 10 analytical condition. condition. Method X: Preparative Conditions: Berger SFC MGII; Method O: Preparative Conditions: Berger SFC MGII; Column: Chiral IC 25x3 cm ID, 5-um: Mobile Phase: 75/25 Column: Chiral OJ 25x3 cm ID, 5-lum; Mobile Phase: 90/10 CO/MeOH w/0.1% diethylamine; Detector Wavelength: CO/MeOH: Detector Wavelength: 220 nm: Flow: 85 220 nm; Flow: 85 mL/min. Analytical Conditions: Aurora mL/min. Analytical Conditions: Aurora analytical SFC: Col 15 analytical SFC: Column: Chiral IC 250x4.6 mm ID, 5um; umn: Chiral OJ 250x4.6 mm ID, 5um; Mobile Phase: 90/10 Mobile Phase: 75/25 CO/MeOH w/0.1% diethylamine: CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the Flow: 2.0 mL/min: Tr corresponds to the analytical condi analytical condition. tion. Method P: Preparative Conditions: Waters SFC-100 MS; Method Y: Mobile Phase: 80/20 CO/MeOH/CAN 50/50: Column: PHENOMENEX(R) LUX Cellulose-225x3 cm ID, Flow: 2.0 mL/min: Tr corresponds: Preparative Conditions: 5 um; Mobile Phase: 75/25 CO/MeOH: Detector Wave Berger SFC MGII: Column: Chiral AD 25x3 cm, 5-um: length: 220 nm, Flow: 100 mL/min. Analytical Conditions: Mobile Phase: 80/20 CO/MeOH/CAN 50/50; Detector Aurora analytical SFC: Column: PHENOMENEXR LUX Wavelength: 220 nm, Flow: 85 mL/min. Analytical Condi Cellulose-2 250x4.6 mm ID, 5 um; Mobile Phase: 75/25 tions: Aurora analytical SFC: Column: Chiral AD 250x4.6 CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the 25 mm ID, 5 um; to the analytical condition. analytical condition. Method Z: Preparative Conditions: Berger SFC MGII; Method Q: Preparative Conditions: Berger SFC MGII; Column: Chiral IC 25x3 cm, 5-lum; Mobile Phase: 83/17 Column: Chiral AD 25x3 cm ID, 5-lum; Mobile Phase: 80/20 CO/MeOH: Detector Wavelength: 220 nm: Flow: 85 CO/MeOH: Detector Wavelength: 220 mm; Flow: 85 mL/min. Analytical Conditions: Aurora analytical SFC: Col mL/min. Analytical Conditions: Aurora analytical SFC: Col 30 umn: Chiral IC 250x4.6 mm ID, 5um; Mobile Phase: 80/20 umn: Chiral AD 250x4.6 mm ID, 5um; Mobile Phase: 80/20 CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the analytical condition. analytical condition. Method AA: Preparative Conditions: Waters SFC100 Method R: Preparative Conditions: Berger SFC MGII; MS: Column: Chiral AS-H coupled Chiral OJ-H 25x3 cm, Column: Chiral AD 25x3 cm ID, 5-lum; Mobile Phase: 87/13 35 5-um: Mobile Phase: 70/30 CO/MeOH: Detector Wave CO/MeOH: Detector Wavelength: 220 mm; Flow: 85 length: 220 nm, Flow: 100 mL/min. Analytical Conditions: mL/min. Analytical Conditions: Aurora analytical SFC: Col AGILENTR) analytical SFC: Column: Chiral AS-H coupled umn: Chiral AD 250x4.6 mm ID, 5um; Mobile Phase: 85/15 to Chiral OJ-H 250x4.6 mm ID, 5um; Mobile Phase: 70/30 CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the analytical condition. 40 analytical condition. Method S: Preparative Conditions: Berger SFC MGII; Method AB: Waters Acquity SDS using the following Column: Chiral IF 25x3 cm ID, 5-um: Mobile Phase: 75/25 method: Linear Gradient of 2% to 98% Solvent B over 1.6 CO/MeOH: Detector Wavelength: 220 mm; Flow: 85 min; UV visualization at 220 nmi; Column: BEH C18 2.1 mL/min. Analytical Conditions: Aurora analytical SFC: Col mmx50mm; 1.7 um particle (Heated to Temp. 50° C.); Flow umn: Chiral IF 250x4.6 mm ID, 5um; Mobile Phase: 70/30 45 rate: 1 ml/min: Mobile Phase A: 100% Water, 0.05% TFA: CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the Mobile Phase B: 100% Acetonitrile, 0.05% TFA. analytical condition. Method AC: Preparative Conditions: Berger SFC MGII; Method T. Preparative Conditions: Waters SFC100-MS; Column: Chiral AD 25x3 cm ID, 5-um: Mobile Phase: 90/10 Column: Chiral IC 25x3 cm ID, 5-lum coupled to WHELK CO/MeOH: Detector Wavelength: 220 nm: Flow: 85 OR. R.R KROMASILR 25x3 cm ID 5-um: Mobile Phase: 50 mL/min. Analytical Conditions: Aurora analytical SFC: Col 70/30 CO/MeOH: Detector Wavelength: 220 nm: Flow: umn: Chiral AD 250x4.6 mm ID, 5um; Mobile Phase: 90/20 100 mL/min. Analytical Conditions: Aurora analytical SFC: CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the Column: Chiral IC 250x4.6 mm ID, 5 um coupled to analytical condition. WHELK-OR. R.R KROMASILR 25x3 cm ID 5-um: Mobile Method AD: Preparative Conditions: Berger SFC MGII; Phase: 70/30 CO/MeOH: Flow: 2.0 mL/min: Tr corre 55 Column: Whelk-O1 Kromasil 25x3 cm ID, 5-lum particles: sponds to the analytical condition. Mobile Phase: 85/15 CO/MeOH: Detector Wavelength: Method U: Preparative Conditions: Waters SFC100-MS; 220 nm; Flow: 85 mL/min. Analytical Conditions: Aurora Column: Chiral OJ-H 25x3 cm ID, 5-um: Mobile Phase: analytical SFC: Column: Whelk-O1 Kromasil 250x4.6 mm 70/30 CO/MeOH: Detector Wavelength: 220 nm: Flow: ID, 5 um; Mobile Phase: 85/15 CO/MeOH: Flow: 2.0 100 mL/min. Analytical Conditions: Aurora analytical SFC: 60 mL/min: Tr corresponds to the analytical condition. Column: Chiral OJ-H 250x4.6 mm ID, 5um; Mobile Phase: Method AE: Preparative Conditions: Berger SFC MGII; 70/30 CO/MeOH: Flow: 2.0 mL/min: Tr corresponds to the Column: Whelk-O1 Kromasil 25x3 cm ID, 5-lum particles: analytical condition. Mobile Phase: 75/25 CO/MeOH: Detector Wavelength: Method V: Preparative Conditions: Berger SFC MGII; 220 nm; Flow: 85 mL/min. Analytical Conditions: Aurora Column: Chiral WHELK-OR 25x3 cm ID, 5-um: Mobile 65 analytical SFC: Column: Whelk-O1 Kromasil 250x4.6 mm Phase: 80/20 CO/MeOH: Detector Wavelength: 220 nm: ID, 5 um; Mobile Phase: 75/25 CO/MeOH: Flow: 2.0 Flow: 85 mL/min. Analytical Conditions: Aurora analytical mL/min: Tr corresponds to the analytical condition. US 9,598.422 B2 53 54 NMR Employed in Characterization of Examples min and then concentrated under reduced pressure. The H NMR spectra (unless otherwise noted) were obtained crude reaction mixture was purified using silica gel chro with JEOL(R) or Bruker FOURIER(R) transform spectrom matography (0% to 100% EtOAc in hexanes) to afford the eters operating at 400 MHz or 500 MHz. desired product. Spectral data are reported as chemical shift (multiplicity, number of hydrogens, coupling constants in HZ) and are reported in ppm (8 units) relative to either an internal CHO standard (tetramethylsilane-0 ppm) for "H NMR spectra, or are referenced to the residual solvent peak (2.49 ppm for CDSOCDH, 3.30 ppm for CDHOD, 1.94 for CHD.CN, 10 7.26 ppm for CHC1, 5.32 ppm for CDHCl). Abbreviations used in the description of NMR peaks: “a”-apparent, “br. s.”-broad singlet. 15 EXAMPLES Cis-4-phenylcyclohexane-1-carbaldehyde: Prepared according the literature procedure (Fox, B. M. et al., J. Med. General Procedures Chem., 57:3464-3483 (2014)). The crude mixture was puri fied using silica gel chromatography (0% to 10% EtOAc in General Procedure A. Amide Bond Formation from Acid. hexane) to afford the desired product as the first eluting isomer.
OH Hess 25 N OH R R
To a stirred solution of carboxylic acid (4.4 mmol) in dimethylformamide (DMF, 15 mL) was added aniline (6.6 30 mmol), diisopropylethylamine (1.53 mL, 8.8 mmol) and 1-bis(dimethylamino)methylene-1H-1,2,3-triazolo 4,5-b] pyridinium 3-oxid hexafluorophosphate (HATU) (2.00 g, 5.28 mmol). The resulting reaction mixture was stirred at rt Cis-(4-phenylcyclohexyl)methanol: To a solution of cis for 3 h, at which point 3 M HCl (30 mL) and CHCl (30 35 4-phenylcyclohexane-1-carbaldehyde (825 mg, 4.4 mmol) mL) were added. The layers were separated, and the aqueous layer was extracted with CHCl (2x30 mL). The combined in THF (25 mL) and MeOH (7 mL) at rt was added NaBH organic extracts were dried over anhydrous Sodium Sulfate portionwise over 5 min. The resulting mixture was stirred at and concentrated under reduced pressure. The resulting rt for 45 min. Then HCl (1 M) was added dropwise. The crude residue was purified by silica gel chromatography to 40 mixture was extracted with EtOAc (3x). The combined afford the desired product(s). organic layers were washed with brine, dried over NaSO General Procedure B. Reaction Between Amines and Acyl and concentrated under reduced pressure. The resulting Chlorides. crude mixture was purified employing silica gel chromatog raphy (0% to 25% EtOAc in hexanes) to afford the desired 45 product.
C Air -- -e- O 50
55 Cis-(4-(iodomethyl)cyclohexyl)benzene: To a solution of cis-(4-phenylcyclohexyl)methanol (2 g, 10.5 mmol), triph enylphosphine (3.3g, 12.6 mmol) and imidazole (1.1 g, 15.8 60 mmol) in CHCl (70 mL) at 0°C. was added iodine (3.5 g. 13.7 mmol). The mixture was warmed to rt and stirred at rt Y for 2 h. The mixture was diluted with CHC1 and washed O with sodium thiosulfate (2M). The organic layer was dried over anhydrous MgSO filtered, and concentrated under To a solution of the amine (1.1 equiv) and NEts (5.0 65 reduced pressure. The crude reaction mixture was purified equiv) in CHCl (0.1 M) was added the acyl chloride (1.0 employing silica gel chromatography (0% to 25% EtOAc in equiv). The resulting reaction mixture was stirred at rt for 15 hexanes) to afford the desired product as an oil (3 g, 95%). US 9,598.422 B2 55 56 2.66-2.59 (m. 1H), 2.06-2.00 (m, 1H), 1.80-1.69 (m, 8H). m/z. 319.3 (M+H"). Example 2
Cis-3-cyano-N-((4-phenylcyclohexyl)methyl)benz amide
Cis-(4-(azidomethyl)cyclohexyl)benzene: To a solution 10 of cis-(4-(iodomethyl)cyclohexyl)benzene (2.6 g., 8.8 mmol) in DMF (44 mL) was added sodium azide (2.8 g., 43.8 mmol). The mixture was stirred at rt for 2 h. Then more sodium azide (1.14 g. 17.5 mmol) was added and the mixture was stirred at rt for 18 h. The mixture was diluted 15 with EtO and washed with water, 1 M LiCl (2x) and brine. CN The organic layers were dried over NaSO and concen trated under reduced pressure to give the desired product (1.5 g., 80%).
Prepared with General Procedure B employing cis-(4- 25 phenylcyclohexyl)methanamine (19 mg, 0.1 mmol), 3-cya nobenzoyl chloride (17 mg, 0.1 mmol), and NEt (51 mg, 0.5 mmol) in CHCl2 (1 mL). Purified using silica gel chroma tography (10% to 30% EtOAc in hexanes) to afford the Cis-(4-phenylcyclohexyl)methanamine: To a solution of 30 desired product as a white solid. "H NMR (400 MHz: cis-(4-(azidomethyl)cyclohexyl)benzene (1.5 g, 7.0 mmol) CDC1): 8 8.09-8.08 (m, 1H), 8.04 (dt, J–7.9, 1.5 Hz, 1H), in THF (35 mL) was added triphenylphosphine (2.56 g. 9.8 7.76 (dt, J–7.7, 1.3 Hz, 1H), 7.56 (t, J–7.8 Hz, 1H), mmol). The mixture was stirred at rt for 30 min and then 7.32-7.17 (m, 5H), 6.49-6.46 (m, 1H), 3.58 (dd, J=7.7, 6.0 water (0.83 mL) was added. The mixture was stirred at rt for Hz, 2H), 2.66-2.59 (m, 1H), 2.07-2.01 (m. 1H), 1.80-1.68 24 h. The mixture was preabsorbed onto silica gel and 35 (m, 8H), m/z. 319.2 (M+H"). purified employing silica gel chromatography 0% to 5% (2 MNH in MeOH) in CHCl to afford the desired product as an oil (1.2g, 94%). Example 3 Example 1 40 Cis-4-chloro-N-(4-phenylcyclohexyl)methyl)benz amide Cis-4-cyano-N-(4-phenylcyclohexyl)methyl)benz amide 45
50
C
CN 55
Prepared with General Procedure B employing cis-(4- phenylcyclohexyl)methanamine (19 mg, 0.1 mmol), 4-chlo Prepared with General Procedure B employing cis-(4- 60 robenzoyl chloride (19 mg, 0.1 mmol), and NEt (51 mg, 0.5 phenylcyclohexyl)methanamine (19 mg, 0.1 mmol), 4-cya mmol) in CHCl2 (1 mL). Purified using silica gel chroma nobenzoyl chloride (17 mg, 0.1 mmol), and NE t (51 mg, 0.5 tography (0% to 20% EtOAc in hexanes) to afford the mmol) in CHCl (1 mL). Purified using silica gel chroma desired product as a white solid. "H NMR (400 MHz: tography (10% to 30% EtOAc in hexanes) to afford the CDC1): 8 7.73-7.69 (m, 2H), 741-7.38 (m, 2H), 7.31-7.23 desired product as a white solid. "H NMR (400 MHz: 65 (m, 4H), 7.20-7.16 (m, 1H), 3.55 (dd, J=7.7, 5.9 Hz, 2H), CDC1): 8 7.89-7.86 (m, 2H), 7.74-7.71 (m, 2H), 7.32-7.17 2.63-2.59 (m. 1H), 2.04-1.98 (m, 1H), 1.79-1.67 (m, 8H). (m, 5H), 6.32-6.30 (m. 1H), 3.58 (dd, J=7.7, 6.0 Hz, 2H), m/z 328.2 (M+H"). US 9,598.422 B2 57 58 Example 4 Example 6 Cis-4-chloro-N-(4-phenylcyclohexyl)methyl)benze Cis-3-chloro-N-(4-phenylcyclohexyl)methyl)benz nesulfonamide amide
10
H C C 15 N A S
Prepared in the manner of General Procedure B employ ing cis-(4-phenylcyclohexyl)methanamine (38 mg, 0.2 mmol), 4-chlorobenzenesulfonyl chloride (42 mg, 0.2 mmol), and NEt (101 mg 0.5 mmol) in CHCl (1 mL). Purified using silica gel chromatography (0% to 25% EtOAc in hexanes) to afford the desired product as a white solid. "H 25 NMR (400 MHz: CDC1): 8 7.87-7.83 (m, 2H), 7.52-7.48 Prepared with General Procedure B employing cis-(4- (m. 2H), 7.31-7.27 (m, 2H), 7.20-7.16 (m,3H), 5.10 (t, J=6.2 phenylcyclohexyl)methanamine (19 mg, 0.1 mmol), 3-chlo HZ, 1H), 3.01 (dd, J=7.7, 6.3 Hz, 2H), 2.56 (dt, J=9.8, 5.0 robenzoyl chloride (19 mg, 0.1 mmol), and NEt (51 mg, 0.5 HZ, 1H), 1.87-1.81 (m. 1H), 1.68-1.52 (m, 8H), m/z. 364.1 mmol) in CHCl (1 mL). Purified using silica gel chroma (M+H"). tography (0% to 20% EtOAc in hexanes) to afford the 30 Example 7 desired product as a white solid. "H NMR (400 MHz: CDC1): 8 7.76 (t, J=1.8 Hz, 1H), 7.64 (dt, J=7.7, 1.4 Hz, (4-Benzylpiperidin-1-yl)(4-chlorophenyl)methanone 1H), 7.47-7.44 (m. 1H), 7.38-7.34 (m. 1H), 7.31-7.23 (m, 4H), 7.20-7.16 (m, 1H), 6.30-6.27 (m. 1H), 3.56 (dd, J=7.7, 6.0 Hz, 2H), 2.64-2.58 (m. 1H), 2.04-1.99 (m. 1H), 1.79 35 1.66 (m, 8H), m/z 328.2 (M+H"). H Example 5 40 N Cis-4-fluoro-N-(4-phenylcyclohexyl)methyl)benz O Ol C amide To 4-chlorophenyl isocyanate (154 mg, 1.0 mmol) in EtO (5 mL) was added 4-benzyl piperidine (193 mg, 1.1 45 mmol) The homogenous reaction mixture produced a pre cipitate over 15 min. The reaction mixture was cooled to 0° C. and the solids were collected by filtration washing with additional EtO (25 mL) to provide the desired product as a white solid. "H NMR (400 MHz: CDC1): 8 7.31-7.28 (m, 50 4H), 7.25-7.21 (m, 3H), 7.16-7.14 (m, 2H), 6.32 (s, 1H), 4.05-4.01 (m, 2H), 2.83 (td, J=12.9, 2.1 Hz, 2H), 2.57 (d. J=6.9 Hz, 2H), 1.77-1.70 (m, 3H), 1.31-1.20 (m, 2H), m/z 329.2 (M+H"). Example 8 55 (4-Benzylpiperidin-1-yl)(3-chlorophenyl)methanone
60 Prepared with General Procedure B employing cis-(4- H phenylcyclohexyl)methanamine (16 mg, 0.1 mmol), 4-fluo robenzoyl chloride (19 mg, 0.1 mmol), and NEt (51 mg, 0.5 N Cl O mmol) in CHCl2 (1 mL). Purified using silica gel chroma 65 tography (0% to 20% EtOAc in hexanes) to afford the desired product as a white solid. US 9,598.422 B2 59 60 To 3-chlorophenyl isocyanate (154 mg, 1.0 mmol) in solution was added 6 mL (95 mmol. 1.25 equiv.) of EtO (5 mL) was added 4-benzyl piperidine (193 mg, 1.1 iodomethane followed by cesium carbonate (43.3 g 133 mmol) The homogenous reaction mixture produced a pre mmol. 1.75 equiv.). This mixture was then stirred for 16 cipitate over 15 min. The reaction mixture was cooled to 0° hours until starting material was consumed as monitored by C. and the solids were collected by filtration washing with additional EtO (25 mL) to provide the desired product as a LCMS. The reaction was then quenched by cooling to 0°C. white solid. "H NMR (400 MHz: CDC1): 8 7.47 (t, J=1.2 and subsequent addition of 1.35 L of water. The mixture was HZ, 1H), 7.29 (q, J=6.4 Hz, 2H), 7.23-7.14 (m, 5H), 701 then extracted with ethyl acetate (3x500 mL) and the com 6.96 (m, 1H), 6.33 (s, 1H), 405-4.01 (m, 2H), 2.83 (td, bined organics were washed with brine (1 L) and dried over J=12.9, 2.1 Hz, 2H), 2.59-2.52 (m, 2H), 1.78-1.71 (m, 3H), sodium sulfate before filtration and concentration. The crude 1.31-1.21 (m, 2H), m/z 329.2 (M+H"). 10 residue was purified via column chromatography (5% ethyl acetate in hexanes) to afford the final compound as a clear Example 9 oil in 69% yield. (R, 0.5 in 10% ethyl acetate in hexanes). Cis-N-4-chloro-(2-(4-(4-methoxyphenyl)cyclohexyl) ethyl)aniline 15 9D. 2-(4-(4-Methoxyphenyl)cyclohexyl)acetic acid Lithium hydroxide (1.58 g. 66.2 mmol) was added to C water (8 mL). The slurry was allowed to stand at rt for 30 min before filtering. The filtrate was added to a solution of the product of Example 9C (2.95 g, 10.67 mmol) in EtOH (9 mL). The slurry was stirred at rt for 2 d and diluted with water. The mixture was filtered and the solid was diluted with EtOAc and 1 MHC1. The layers were separated and the NH 25 organic layer was dried over Sodium sulfate and concen trated under reduced pressure to provide 2-(4-(4-methoxy N phenyl)cyclohexyl)acetic acid.
30 9E and 9F. cis-N-(4-Chlorophenyl)-2-(4-(4- 9A. Ethyl methoxyphenyl)cyclohexyl)acetamide and trans-N- 2-(4-(4-hydroxyphenyl)cyclohexylidene)acetate (4-Chlorophenyl)-2-(4-(4-methoxyphenyl)cyclo Triethylphosphonoacetate hexyl)acetamide
(46.9 mL, 236 mmol) in THF (250 mL) was added 35 Prepared with General Procedure A employing 2-(4-(4- dropwise over 1 hour at 0°C. to a solution of NaH (60% methoxyphenyl)cyclohexyl)acetic acid (product of Example dispersion in oil, 11.8 g. 295 mmol) in THF (120 mL). The 9D, 124 mg. 0.5 mmol), 4-chloroaniline (97 mg, 0.75 mixture was warmed to rt and stirred at rt for 1 h. In a mmol), HATU (435 mg, 0.75 mmol), and PrNEt (323 mg, separate flask, a solution of 4-(4-hydroxyphenyl)cyclo 2.5 mmol) in DMF (1.0 mL). Purification using silica gel hexanone (37.5 g., 197 mmol) in THF (250 mL) was added 40 chromatography (0% to 25% EtOAc in hexanes) afforded carefully to a mixture of NaH (60% dispersion in oil, 8.67 cis-N-(4-chlorophenyl)-2-(4-(4-methoxyphenyl)cyclo g, 216 mmol) in THF (100 mL) at 0°C. The mixture was hexyl)acetamide (Example 9E), a white solid, as the first stirred at rt for 2 hours. The mixture of the cyclohexanone eluting isomer and trans-N-(4-chlorophenyl)-2-(4-(4- was added to the phosphonate mixture at 0° C. via cannu lation. The mixture was warmed to rt and stirred at rt for 2 methoxyphenyl)cyclohexyl)acetamide (Example 9F) as the h. The mixture was quenched by careful addition of ice and 45 second eluting isomer. water (1 L) and subsequently extracted with ethyl acetate (3x500 mL) and the combined organics were then washed cis-N-(4-Chlorophenyl)-2-(4-(4-methoxyphenyl) with brine (1 L), dried over sodium sulfate, filtered, and cyclohexyl)acetamide concentrated to provide ethyl 2-(4-(4-hydroxyphenyl)cyclo hexylidene)acetate in 97% yield as a white solid. 50 H NMR (400 MHz: CDC1): 8 7.49-7.45 (m, 2H), 9B. Ethyl 2-(4-(4-hydroxyphenyl)cyclohexyl)acetate 7.29-7.26 (m, 2H), 7.17-7.15 (m, 3H), 6.87-6.83 (m, 2H), 3.79 (s.3H), 2.63-2.55 (m, 1H), 2.45-2.37 (m, 3H), 1.77 To a solution ethyl 2-(4-(4-hydroxyphenyl)cyclohex 1.64 (m, 8H), m/z. 358.2 (M+H"). ylidene)acetate (9.74 g., 35.8 mmol) in ethyl acetate was 55 added Pd/C (0.974 g. 10 wt.%). The reaction solution was sparged with a balloon of H gas and stirred overnight under C an atmosphere of hydrogen for 2 days. The reaction mixture was filtered through CELITE(R), washing generously with ethyl acetate, and concentrated under reduced pressure to afford the desired product as a white crystalline solid in 60 quantitative yield as a mixture of diastereomers. 9C. Ethyl NH 2-(4-(4-methoxyphenyl)cyclohexyl)acetate 65 A solution of the product of Example 9B (20.0 g, 76.2 mmol. 1.0 equiv.) was dissolved in 770 mL of DMF. To this US 9,598.422 B2 61 62 Example 9 11 A. (+/-)-Cis-3-phenylcyclopentan-1-ol Cis-N-4-chloro-(2-(4-(4-methoxyphenyl)cyclohexyl) ethyl)aniline 3-Phenylcyclopentan-1-one was prepared as previously described (Yamamoto, T. et al., J. Organomet. Chem., 694: To a solution of cis-N-(4-chlorophenyl)-2-(4-(4-methoxy 1325-1332 (2009)). To a solution of 3-phenylcyclopentan phenyl)cyclohexyl)acetamide (33 mg 0.092 mmol) in THF 1-one (1.0 g. 6.2 mmol) in 30 mL of methanol cooled to 0° (0.5 mL) at rt was added Borane tetrahydrofuran complex C. was added NaBH (0.27g, 7.2 mmol). The ice bath was solution (0.5 mL, 0.5 mmol. 1 M in THF). The resulting removed, and the reaction was allowed to warm to rt and mixture was stirred for 2.5 h at rt at which point aqueous 10 stirred for 3 h. The reaction was quenched with 1M HCl and HCl (1 M) was added and the mixture was stirred at rt for diluted with EtOAc (30 mL), and the layers were separated. 30 min. Evolution of gas was observed. The mixture was The organic extracts were dried over anhydrous MgSO, basified with sat. NaCO and extracted with CHCl (2x). filtered, and concentrated under reduced pressure. The The combined organic layers were dried over anhydrous resulting crude mixture of ~1.6:1 cis: trans alcohols was Na2SO, filtered, and concentrated under reduced pressure. purified using silica gel chromatography (15% EtOAC in The resulting crude mixture was purified employing silica 15 gel chromatography (0% to 10% EtOAc in hexanes) to pentane) to afford the desired cis-3-phenylcyclopentan-1-ol afford the desired product. "H NMR (400 MHz, CDC1) & as a colorless oil (118 mg, 12%). H NMR (400 MHz: 7.19-7.09 (m, 4H), 6.90-6.81 (m, 2H), 6.56 (d. J=8.7 Hz, CDC1): 8 7.16-7.09 (m, 5H), 4.43-440 (m. 1H), 3.06-3.00 2H), 3.80 (s, 3H), 3.17-3.07 (m, 2H), 2.55 (s, 1H), 1.85 (s, (m. 1H), 2.70 (brs, 1H), 2.53-2.43 (m. 1H), 2.06-1.63 (m, 1H), 1.80-1.63 (m, 10H). m/z 344.2 (M+H"). 5H). Example 10 Example 11 Trans-N-4-chloro-(2-(4-(4-methoxyphenyl)cyclo hexyl)ethyl)aniline 25 (+/-)-Cis-3-phenylcyclopentyl(4-chlorophenyl)car bamate To a solution of cis-3-phenylcyclopentan-1-ol (150 mg, C 0.95 mmol) in CHCH was added 4-chlorophenyl isocya 30 nate (150 mg, 0.95 mmol). After 5 min, the reaction mixture was concentrated under reduced pressure and triturated with diethyl ether (10 mL). The mixture was filtered, rinsing with diethyl ether, to afford the desired product as a white solid. NH "H NMR (400 MHz: DMSO-d): 8 9.79 (brs, 1H), 7.49 (d. t J=7.5 Hz, 2H), 7.33-7.14 (m, 7H), 5.09-5.04 (m. 1H), 3.13-3.02 (m, 1H), 2.60-2.48 (m, 1H), 2.08-1.61 (m, 6H). NO Example 12 Prepared using the procedure from the previous example employing 73 mg of trans-N-(4-chlorophenyl)-2-(4-(4- 40 methoxyphenyl)cyclohexyl)acetamide. Purified using silica Cis-(4-phenylcyclohexyl)methyl(4-fluorophenyl) gel chromatography (0% to 10% EtOAc in hexanes) to carbamate afford the desired product. H NMR (400 MHz, CDC1) & 7.17-7.09 (m, 4H), 6.89-6.81 (m, 2H), 6.58-6.51 (m, 2H), 3.80 (s, 3H), 3.18-3.09 (m, 2H), 2.45 (t, J=12.3 Hz, 1H), 45 1.90 (d. J–12.3 Hz, 4H), 1.67-1.47 (m, 4H), 1.44 (dd. J=17.5, 7.8 Hz, 2H), 1.14 (dd, J=22.1, 12.0 Hz, 2H), m/z 344.2 (M+H"). Example 11 50 (+/-)-Cis-3-phenylcyclopentyl(4-chlorophenyl)car O bamate y 55 O
C To a solution of cis-(4-phenylcyclohexyl)methanol (200 mg, 1.05 mmol) in diethyl ether (5 mL) was added 4-fluo 60 rophenyl isocyanate (144 mg, 1.05 mmol). Upon consump tion of the starting materials, the resulting Solution was concentrated to provide a white solid, which was triturated in diethyl ether (3 mL) and filtered to afford the desired y product as a white solid. "H NMR (400 MHz: DMSO-d): 65 & 9.65 (brs, 1H), 748-7.42 (m, 2H), 7.18-7.06 (m, 7H), 4.20 (d. J=9.5 Hz, 2H), 2.61-2.51 (m. 1H), 2.07-2.01 (m. 1H), 1.77-1.57 (m, 8H). US 9,598.422 B2 63 64 Example 13 Example 15
Trans-1-(4-fluorophenyl)-3-(4-phenylcyclohexyl) Trans-1-(3-chlorophenyl)-3-(4-phenylcyclohexyl) la la
10 C
15 H H N N NH '' t y O O
To a stirred solution of trans-4-phenylcyclohexylamine (Combi-Blocks, San Diego, Calif.) (50 mg, 0.3 mmol) in 25 To a stirred solution of trans-4-phenylcyclohexylamine diethyl ether was added 4-fluorophenyl isocyanate (0.032 (50 mg, 0.3 mmol) in diethyl ether (1.4 mL) was added mL, 0.3 mmol) at rt. After stirring for 30 min the voluminous 3-chlorophenyl isocyanate (0.035 mL, 0.3 mmol) at rt. After white precipitate was isolated by vacuum filtration to yield stirring for 30 min the voluminous white precipitate was the desired product. "H NMR (400 MHz, CDC1) & 7.35 isolated by vacuum filtration and concentrated under 7.14 (m, 7H), 7.07-6.97 (m, 2H), 6.00 (s, 1H), 4.41-4.30 (m, 30 reduced pressure to yield the desired product. H NMR (400 1H), 3.83-3.65 (m. 1H), 2.56-2.39 (m. 1H), 2.21-2.10 (m, MHz, CDC1) & 7.42 (t, J–2.0 Hz, 1H), 7.34-7.11 (m, 7H), 2H), 1.99-1.88 (m, 2H), 1.72-1.46 (m, 2H), 1.37-1.07 (m, 7.08-7.02 (m, 1H), 6.14 (s, 1H), 4.47 (s, 1H), 3.85-3.62 (m, 2H). 1H), 2.61-2.39 (m. 1H), 2.27-2.12 (m, 2H), 2.02-1.89 (m, 35 2H), 1.74-1.57 (m, 2H), 1.39-1.19 (m, 2H). Example 14 Example 16 Trans-1-(4-chlorophenyl)-3-(4-phenylcyclohexyl) la 40 Trans-2-(3-chlorophenyl)-N-(4-phenylcyclohexyl) acetamide
45 C
C
50 H Ny O 55
To a stirred solution of trans-4-phenylcyclohexylamine Prepared according to General Procedure A using trans (50 mg 0.3 mmol) in diethyl ether was added 4-chlorophe 60 4-phenylcyclohexylamine and 3-chlorophenylacetic acid. nyl isocyanate (44 mg. 0.3 mmol) at rt. After stirring for 30 Purified by silica gel chromatography (0% to 50% ethyl min the voluminous white precipitate was isolated by acetate in hexanes) which afforded the desired product as vacuum filtration to yield the desired product. H NMR (400 white solid. "H NMR (400 MHz, CDC1) & 7.36-7.12 (m, MHz, CDC1) & 7.38-7.12 (m, 9H), 6.05 (s, 1H), 4.39 (d. 9H), 5.21 (d. J=7.9 Hz, 1H), 3.84 (tdt, J=12.0, 8.1, 4.0 Hz, J–7.4 Hz, 1H), 3.88-3.61 (m. 1H), 2.56-2.40 (m. 1H), 65 1H), 3.53 (s. 2H), 2.51-2.37 (m, 1H), 2.13-1.99 (m, 2H), 2.28-2.12 (m, 2H), 2.00-1.89 (m, 2H), 1.71-1.59 (m, 2H), 1.99-1.85 (m, 2H), 1.67-1.49 (m, 2H), 1.29-1.09 (m, 2H). 1.36-1.16 (m, 2H). m/z 328.2 (M+H"). US 9,598.422 B2 65 66 Example 17 Example 19 Cis-2-(4-chlorophenyl)-N-(4-phenylcyclohexyl)acet Cis-1-(4-chlorophenyl)-3-(4-phenylcyclohexyl)urea amide
C
C 10
H N
H 15 Ny-l O Prepared according to General Procedure A using cis-4- phenylcyclohexylamine and 4-chlorophenylacetic acid (Li, G. et al., Bioorg. Med. Chem. Lett., 18:1146-1150 (2008)). Purified by silica gel chromatography (0% to 50% ethyl acetate in hexanes) which afforded the desired product as To a stirred solution of cis-4-phenylcyclohexylamine (Li, white solid. "H NMR (400 MHz, CDC1) & 743-7.17 (m, 25 7H), 7.07 (dd, J-7.5, 0.8 Hz, 2H), 5.86 (s, 1H), 4.25-4.05 (m, G. et al., Bioorg Med. Chem. Lett., 18:1146-1150 (2008)) 1H), 3.60 (s. 2H), 2.60-2.46 (m, 1H), 1.89-1.55 (m, 6H), (60 mg. 0.34 mmol) in diethyl ether (1.4 mL) was added 1.42-1.22 (m, 2H), m/z 328.2 (M+H"). 4-chlorophenyl isocyanate (53 mg, 0.34 mmol) at rt. After General Procedure C. Reaction Between Esters and Ani stirring for 30 min the voluminous white precipitate was lines. isolated by vacuum filtration and concentrated under 30 reduced pressure to yield the desired product. H NMR (400 MHz, CDC1) & 7.37-7.09 (m, 9H), 6.28 (s, 1H), 4.88 (d. R Air R A. OEt A NH J–7.2 Hz, 1H), 4.17-4.03 (m. 1H), 2.68–2.49 (m. 1H), HN He 1.98-1.87 (im, 2H), 1.87-1.69 (m, 4H), 1.65-1.50 (m, 2H). 35 O O
Example 18 To a solution of the aniline (2.0 equiv) in THF (0.25 M) at 0°C. was added a solution of PrMgCl (2.0 equiv, 2 M in THF). The resulting solution was warmed to rt and stirred Cis-1-(3-chlorophenyl)-3-(4-phenylcyclohexyl)urea 40 for 5 min at which point the ester (1.0 equiv) was added dropwise. The resulting reaction mixture was stirred at rt for 8 h and was poured into water. EtOAc was added, and the layers were separated. The aqueous layer was extracted with EtOAc (3x). The combined organic extracts were dried over 45 anhydrous MgSO filtered, and concentrated under reduced pressure. The crude reaction mixture was purified using C silica gel chromatography (0% to 100% EtOAc in hexanes) to afford the desired product(s). 50 Example 20 H N Cis-4-benzyl-N-(4-chlorophenyl)cyclohexane-1- y carboxamide O 55
To a stirred solution of cis-4-phenylcyclohexylamine (60 C mg, 0.36 mmol) in diethyl ether (1.4 mL) was added 3-chlorophenyl isocyanate (0.042 mL, 0.34 mmol) at rt. After stirring for 30 min the voluminous white precipitate 60 was isolated by vacuum filtration and concentrated under reduced pressure to yield the desired product. "H NMR (400 MHz, CDC1) & 747-7.42 (m, 1H), 7.35-7.14 (m, 7H), 7.04 NH (dt, J=7.5, 1.7 Hz, 1H), 6.37 (s, 1H), 4.98 (d. J=6.6 Hz, 1H), 65 O 4.21-4.02 (m. 1H), 2.68-2.50 (m, 1H), 2.00-1.87 (im, 2H), 1.88-1.67 (m, 4H), 1.67-1.49 (m, 2H). US 9,598.422 B2 67 68 Prepared using General Procedure C employing ethyl Example 23 4-benzylcyclohexane-1-carboxylate (250 mg, 1.0 mmol), which can be prepared by methods shown in Trans-4-benzyl-N-(4-cyanophenyl)cyclohexane-1- WO2005080317A2, and 4-chloroaniline (191 mg, 1.5 carboxamide mmol). Purification using silica gel chromatography (0% to 5 50% EtOAc in hexanes) afforded the desired product. "H NMR (400 MHz: CDC1): 8 7.49 (d. J=8.8 Hz, 2H), 7.28 (d. CN J=8.7 Hz, 2H), 7.23-7.10 (m, 5H), 2.62 (d. J=7.7 Hz, 2H), 2.48-2.37 (m. 1H), 2.04-1.93 (m, 2H), 1.88-1.82 (m, 2H), 1.74-1.43 (m, 4H), 1.10-0.93 (m, 1H), m/z 328.1 (M+H"). "
Example 21 NH
Trans-4-benzyl-N-(4-chlorophenyl)cyclohexane-1- 15 O carboxamide Further elution from the column in the previous example afforded the desired product as the second eluting isomer. H 2O NMR (400 MHz: CDC1): 8 7.73-7.63 (m, 2H), 7.63-7.55 C (m. 2H)), 7.46 (s, 1H), 7.32-7.24 (m, 2H), 7.23-7.10 (m, 3H), 2.52 (d. J=7.1 Hz, 2H), 2.21 (tt, J=12.1, 3.4 Hz, 1H), 2.04-191 (m, 2H), 1.89-1.77 (m, 2H), 1.74-1.46 (m, 3H), 1.03 (qd, J=13.2, 3.5 Hz, 2H).; m/z. 319.2 (M+H"). 25 Example 24 NH Cis-4-benzyl-N-(4-fluorophenyl)cyclohexane-1- O carboxamide 30 Further elution from the column in the previous example F afforded the desired product as the second eluting isomer. "H NMR (400 MHz. CDC1): 8 7.46 (d. J=8.8 Hz, 2H), 7.31 7.26 (m, 3H), 7.26-7.24 (m. 1H), 7.22-7.11 (m, 4H), 2.52 (d. 35 J=7.1 Hz, 2H), 2.20-2.10 (m, 1H), 1.97 (d. J=11.4 Hz, 2H), 1.86-1.73 (m, 2H), 1.57-1.45 (m, 3H), 1.38-1.24 (m, 2H): m/z 328.1 (M+H"). NH O Example 22 40 Prepared using General Procedure C employing ethyl Cis-4-benzyl-N-(4-cyanophenyl)cyclohexane-1-car 4-benzylcyclohexane-1-carboxylate (250 mg, 1.0 mmol) and 4-fluoroaniline (0.15 mL, 1.5 mmol). Purification using boxamide silica gel chromatography (0% to 50% EtOAc in hexanes) 45 afforded the desired product. "H NMR (400 MHz: CDC1): 8 7.48 (ddd, J=10.5, 6.9, 4.8 Hz, 2H), 7.34-7.11 (m, 5H), 7.09-6.94 (m, 3H), 2.62 (d. J–7.6 Hz, 2H), 2.48-2.36 (m, CN 1H), 2.04-1.92 (m, 2H), 1.88-1.76 (m. 1H), 1.74-1.40 (m, 50 5H), 1.12–0.94 (m. 1H); m/z. 312.2 (M+H"). Example 25 Trans-4-benzyl-N-(4-fluorophenyl)cyclohexane-1- NH carboxamide 55
F Prepared using General Procedure C employing ethyl 4-benzylcyclohexane-1-carboxylate (250 mg, 1.0 mmol) 60 and 4-aminobenzonitrile (177 mg, 1.5 mmol). Purification using silica gel chromatography (0% to 50% EtOAc in hexanes) afforded the desired product. "H NMR (400 MHz: NH CDC1): 8 7.73-7.63 (m, 2H), 7.63-7.55 (m, 2H), 7.51 (s, 1H), 7.32-7.24 (m, 2H), 723-7.10 (m, 3H), 2.61 (d. J=7.6 65 O Hz, 2H), 2.49-2.45 (m, 1H), 2.04-1.91 (m. 1H), 1.89-1.77 (m. 1H), 1.74-1.46 (m, 7H); m/z. 319.2 (M+H"). US 9,598.422 B2 69 70 Further elution from the column in the previous example Hz, 2H), 1.55-1.49 (m, 3H), 1.04 (qd, J=13.3, 3.3 Hz, 2H): afforded the desired product as the second eluting isomer. H m/z 324.2 (M+H"). NMR (400 MHz: CDC1): 8 7.46 (dd, J=9.0, 4.8 Hz, 2H), 7.33-7.11 (m, 5H), 7.10-6.95 (m, 3H), 2.52 (d. J–7.0 Hz, Example 29 2H), 2.20-2.11 (m, 1H), 1.97 (d. J=10.4 Hz, 2H), 1.84 (d. J=13.0 Hz, 2H), 1.61-1.44 (m, 3H), 1.00 (dd, J-29.4, 10.9 Hz, 2H); m/z. 312.2 (M+H"). N-Benzyl-2-(4-(4-methoxyphenyl)cyclohexyl)acet amide
Example 26 10
Cis-4-benzyl-N-(4-methoxyphenyl)cyclohexane-1- carboxamide 15
To solution of 2-(4-(4-methoxyphenyl)cyclohexyl)acetic acid (product of 9D, 152 mg, 0.61 mmol) in CHCl (1.2 mL) at rt was added oxalyl chloride (63 uL, 0.73 mmol) and NH 25 one drop of DMF. Evolution of gas was observed and the mixture turned yellow in color. The mixture was stirred at rt for 1 h and then concentrated under reduced pressure. The residue was dissolved in CHCl (1.2 mL) and benzyl amine (67 uL, 0.61 mmol) and triethylamine (85 uL, 0.61 mmol) Prepared using General Procedure C employing ethyl 30 were added at rt. A white precipitate formed and more 4-benzylcyclohexane-1-carboxylate (250 mg, 1.0 mmol) triethylamine (170 uL. 1.22 mmol) and CHCl (1.2 mL) and 4-methoxyaniline (185 mg, 1.5 mmol). Purification were added. The homogenous mixture was stirred at rt for 3 using silica gel chromatography (0% to 50% EtOAc in h. The mixture was concentrated under reduced pressure. hexanes) afforded the desired product. H NMR (400 MHz: 35 The residue was dissolved in EtOAc and washed with sat. CDC1): 8 7.46-7.38 (m, 2H), 7.33-7.23 (m, 2H), 7.22-7.09 NaHCO, and brine. The organic layer was dried over (m, 4H), 6.92-6.80 (m, 2H), 3.79 (s.3H), 2.62 (d. J=7.6 Hz, 2H), 2.48-2.35 (m, 1H), 2.05-1.95 (m, 2H), 1.86-1.75 (m, Sodium Sulfate and concentrated under reduced pressure. 1H), 1.70-1.63 (m, 2H), 1.65-1.48 (m, 4H); m/z 324.2 The residue was purified using silica gel chromatography (M+H"). 40 (35% EtOAc in hexanes) to afford a mixture of isomers. The residue was recrystallized from heptane/IPA to afford the desired product as white Solid as a 2:1 mixture of trans:cis Example 27 isomers. m/z 338.3 (M+H"). Trans-4-benzyl-N-(4-methoxyphenyl)cyclohexane 45 Example 30 1-carboxamide Cis-N-benzyl-2-(4-(4-methoxyphenyl)cyclohexyl) acetamide 50
55
NH NH
NO O 60 Further elution from the column in the previous example The mother liquors from the previous example were afforded the desired product as the second eluting isomer. H concentrated under reduced pressure to give the desired NMR (400 MHz: CDC1): 8 7.40 (d. J=9.0 Hz, 2H), 7.29 (d. product. "H NMR (400 MHz, CDC1) & 7.45-7.23 (m, 5H), J=7.0 Hz, 2H), 7.23-7.09 (m, 3H), 7.01 (s, 1H), 6.84 (d. 65 7.20-7.07 (m, 2H), 6.92-6.71 (m, 2H), 5.80 (s, 1H), 4.46 (d. J=8.9 Hz, 2H), 3.78 (s.3H), 2.52 (d. J=6.9 Hz, 2H), 2.15 (tt, J=5.7 Hz, 2H), 3.79 (s.3H), 2.66-2.48 (m, 1H), 2.38-2.28 J=12.2, 3.5 Hz, 1H), 1.98 (d. J=11.2 Hz, 2H), 1.83 (d. J=13.6 (m, 3H), 1.79-1.58 (m, 8H); m/z 338.2 (M+H"). US 9,598.422 B2 71 72 Example 31 Example 31 N-(4-Chlorophenyl)-4-phenoxypiperidine-1-carbox N-(4-Chlorophenyl)-4-phenoxypiperidine-1-carbox amide amide To a solution of N-(4-chlorophenyl)-4-hydroxypiperi dine-1-carboxamide (100 mg, 0.39 mmol) and PPhs (430 mg, 1.6 mmol) in THF (2 mL) at rt was added diethyl C aZodicarboxylate (DEAD) (0.068 mL, 0.43 mmol) and phe 10 nol (41 mg 0.43 mmol). The solution was allowed to stir at rt for 16 h before concentrating under reduced pressure. The crude residue was purified using silica gel chromatography (30% EtOAc in hexanes) to afford the desired product as a N clear, colorless film. "H NMR (400 MHz: CDC1): 8 7.34 15 7.22 (m, 6H), 6.99-6.90 (m, 3H), 6.48 (brs, 1H), 4.59-4.54 y (m. 1H), 3.75-3.67 (m, 2H), 3.52-3.46 (m, 2H), 2.4-1.97 (m, O 2H), 1.94-1.86 (m, 2H); m/z 331.2 (M+H"). Example 32 31 A. N-(4-Chlorophenyl)-4-oxopiperidine-1-carboxamide N-(4-Chlorophenyl)-4-phenoxycyclohexane-1-car boxamide Piperidin-4-one hydrochloride (1.37 g, 10.1 mmol) was dissolved in CHCl, washed with 1 M NaOH (60 mL), 25 dried over anhydrous MgSO, filtered, and concentrated C under reduced pressure to provide the free base as a clear, colorless oil. The piperidin-4-one was diluted with CHCl O (6 mL), and the solution was cooled to 0°C. 4-Chlorophenyl isocyanate (1.59 g, 10.1 mmol) was added to the solution, and the ice bath was immediately removed. After 3 h, the 30 reaction mixture was diluted with brine (10 mL) and 1 M NH NaOH (2 mL) and extracted with CHCl (2x30 mL). The combined organic layers were dried over anhydrous MgSO4, O filtered, and concentrated under reduced pressure to afford the desired intermediate as a white solid. "H NMR (400 35 MHz: CDC1): 8.80 (s, 1H), 7.50 (d. J=9.0 Hz, 2H), 7.27 (d. J=8.8 Hz, 2H), 3.72 (t, J=6.2 Hz, 4H), 2.38 (t, J=6.2 Hz, 4H); 32A. N-(4-Chlorophenyl)-4-hydroxycyclohexane-1- m/z 253.1 (M+H"). carboxamide 40 4-Hydroxycyclohexane-1-carboxylic acid (2.0 g, 14 31B. N-(4-Chlorophenyl)-4-hydroxypiperidine-1- mmol), 4-chloroaniline (1.8 g., 14 mmol), and 1-bis(dim carboxamide ethylamino)methylene)-1H-1,2,3-triazolo 4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (6.3, 17 mmol) were To a solution of N-(4-chlorophenyl)-4-oxopiperidine-1- added to a 100 mL round bottom flask followed by DMF (46 carboxamide (401 mg, 1.58 mmol) in methanol (20 mL) was 45 mL) and diisopropylethylamine (2.5 mL, 28 mmol). The added NaBH (89 mg, 2.36 mmol) at rt. The reaction was solution was stirred under argon for 16 h. The reaction allowed to stir for 14 h before adding 1 M HCl (20 mL). The solution was diluted with EtOAc (60 mL), washed with 1 N solution was extracted with CHCl (60 mL), dried over NaOH (50 mL), dried over anhydrous MgSO filtered, and anhydrous MgSO filtered, and concentrated under reduced concentrated under reduced pressure. The crude residue was pressure to afford the desired product as an oil. H NMR 50 purified using silica gel chromatography (0% to 100% (400 MHz: CDC1): 8.57 (s, 1H), 7.46 (d. J=9.0 Hz, 2H), EtOAc in hexanes) to afford the desired product as a white 7.24 (d. J=9.0 Hz, 2H), 4.70 (d. J=4.3 Hz, 1H), 3.79 (td, solid, m/z 254.2 (M+H"). J–4.3, 13.6 Hz, 2H), 3.68-3.58 (m, 1H), 3.02 (ddd, J=3.2. 10.0, 13.3 Hz, 2H), 1.75-1.67 (m, 2H), 1.34-1.21 (m, 2H). Example 32 55 N-(4-Chlorophenyl)-4-phenoxycyclohexane-1-car C boxamide To a 50 mL round bottom flask was added N-(4-chloro 60 phenyl)-4-hydroxycyclohexane-1-carboxamide (1.6 g. 6.3 mmol), polymer-bound PPhs (3.0 mmol/g PPh, 8.4g, 25 mmol), and phenol (0.894 g, 9.5 mmol). The flask was evacuated and backfilled with argon. To the flask was added y THF (30 mL), and the mixture was cooled to 0°C. DEAD 65 (1.49 mL, 9.5 mmol) was added dropwise by syringe, and the ice bath was removed. The mixture was allowed to warm to rt and stirred for 16 h. The reaction mixture was diluted US 9,598.422 B2 73 74 with EtOAc (50 mL), filtered through a pad of 1:1 romethane) to furnish the desired product, (trans)-4-(4- CELITER):silica gel, and concentrated under reduced pres methoxyphenyl)cyclohexan-1-amine, as an off-white solid. Sure. The crude residue was purified using silica gel chro matography (0% to 18%, then 18% to 30% EtOAc in Example 33 hexanes) to afford the desired product as a white solid. "H 5 NMR (400 MHz: CDC1): 8 7.48 (d. J=8.7 Hz, 2H), 7.32 2-(4-Chlorophenyl)-N-((trans)-4-(4-methoxyphenyl) 7.27 (m, 4H), 7.15 (brs, 1H), 6.99-6.84 (m, 3H), 4.34-4.14 cyclohexyl)acetamide (m. 1H), 2.36-2.19 (m, 3H), 2.08 (d. J=11.7 Hz, 2H), 1.81-1.68 (m, 2H), 1.55-1.43 (m, 2H); m/z 330.2 (M+H"). A solution containing 4-chlorophenylacetic acid (109 mg. 10 0.64 mmol) and 1-bis(dimethylamino)methylene)-1H-1.2. Example 33 3-triazolo 4,5-b]pyridinium 3-oxid hexafluorophosphate (266 mg, 0.70 mmol) in DMF (6 mL) was stirred at rt for 10 2-(4-Chlorophenyl)-N-((trans)-4-(4-methoxyphenyl) minutes before (trans)-4-(4-methoxyphenyl)cyclohexan-1- cyclohexyl)acetamide amine (131 mg, 0.64 mmol) was added. After stirring for 20 15 minutes, N,N-diisopropylethylamine (0.33 mL, 1.92 mmol) was added, and the mixture stirred for an additional 1 h. The flask contents were then poured into brine (30 mL) and C filtered. The filtrate was concentrated under reduced pres Sure to afford a residue which was purified using silica gel chromatography (5% MeOH in CHCl2) to yield the desired 2-(4-chlorophenyl)-N-((trans)-4-(4-methoxyphenyl)cyclo hexyl)acetamide as a white solid. "H NMR (400 MHz: H CDC1): 8 7.33 (d. J=8.4 Hz, 2H), 7.21 (d. J=8.1 Hz, 2H), N 7.09 (d. J=8.7 Hz, 2H), 6.83 (d. J=9 Hz, 2H), 5.18 (d. J=8.4 25 HZ, 1H), 3.85-3.78 (m, 4H), 3.52 (s. 2H), 2.43-2.34 (m, 1H), 2.05-2.00 (m, 2H), 1.90-1.85 (m, 2H), 1.51-1.04 (m, 4H) O ppm. m/z. 358.2 (M+H)".
30 Example 34 4-Fluoro-N-(1,1,1-trifluoro-3-(4-(4-methoxyphenyl) cyclohexyl)propan-2-yl)aniline, HCl
33A. cis-4-(4-Methoxyphenyl)cyclohexyl 35 methanesulfonate To a solution of cis-4-(4-methoxyphenyl)-cyclohexanol (Chem. Commun., 48:9376 (2012)) (366 mg, 1.77 mmol) and triethylamine (0.49 mL, 3.55 mmol) in tetrahydrofuran 40 (9 mL), at 0°C., was added methanesulfonyl chloride (0.21 mL, 2.66 mmol). The mixture was stirred for 1.5 h before being quenched with water, diluted with EtOAc, then NH HC washed sequentially with dilute HCl, saturated aqueous Solution of Sodium bicarbonate, and brine. The organic 45 phases were dried over sodium sulfate then concentrated under reduced pressure before the resultant residue was purified using silica gel chromatography (20% to 50% EtOAc in hexanes) to afford cis-4-(4-methoxyphenyl)cyclo hexyl methanesulfonate, as a white Solid. 50 33B. (trans)-4-(4-Methoxyphenyl)cyclohexan-1-amine 34A. Ethyl 2-(4-(4-hydroxyphenyl)cyclohexylidene)acetate To a mixture of cis-4-(4-methoxyphenyl)cyclohexyl 55 methanesulfonate (430 mg, 1.51 mmol) in DMF (7.5 mL) To an oven-dried flask (Flask #1) was added NaH (60% was added sodium azide (108 mg, 1.66 mmol). The mixture dispersion in oil, 11.8 g. 295 mmol) and 120 mL of THF, was then heated at 70° C. for 4 h. The mixture was cooled before the mixture was cooled to 0°C. To this mixture was to rt, quenched with water, then diluted with EtOAc. The added dropwise, over 1 hour, a mixture of triethylphospho organic phase was washed several times with water, then 60 noacetate (46.9 mL, 236 mmol) in 250 mL of THF. After the brine, before being concentrated under reduced pressure to addition was complete, the mixture was stirred for 1 h at rt. ~10 mL. To this mixture was added wet 10% Pd/C (70 mg. To a separate flask (Flask #2), containing a 0°C. mixture 10% w/w) and the reaction vessel was placed under a of NaH (60% dispersion in oil, 8.67 g. 216 mmol) in 100 mL hydrogen atmosphere, at rt, for 16 h. The mixture was THF was carefully added, over 45 minutes, a solution of filtered and the filtrate was concentrated under reduced 65 37.47 g (196.9 mmol) of 4-(4-hydroxyphenyl)cyclo pressure to afford a residue which was purified using silica hexanone in 250 mL THF. After addition was complete, the gel chromatography (10% to 20% methanol in dichlo mixture was stirred at rt for 2 huntil the mixture became a US 9,598.422 B2 75 76 clear solution. Once this solution was clear, Flask #1 was hexanes) to furnish. 2-(4-(4-methoxyphenyl)cyclohexyl)ac cooled to 0° C. and the contents of Flask #2 are added via etaldehyde (609 mg, 52%) as a colorless oil. cannulation. After this addition was complete, the mixture was warmed to rt and stirred for 2 h. The mixture was then 34F. 1,1,1-Trifluoro-3-(4-(4-methoxyphenyl)cyclo quenched by careful addition of ice and water (1 L) and hexyl)propan-2-ol subsequently extracted with EtOAc (3x500 mL). The com bined organic layers were then washed with brine (1 L), A solution of 2-(4-(4-methoxyphenyl)cyclohexyl)acetal dried over sodium sulfate, filtered, and concentrated under dehyde (34E, 609 mg, 2.62 mmol) and TMS-CF (0.58 mL, reduced pressure to provide ethyl 2-(4-(4-hydroxyphenyl) 3.93 mmol) in THF (6 mL) was treated with a 1M solution cyclohexylidene)acetate in 97% yield as a white solid. 10 of TBAF in THF (15.72 mL, 15.72 mmol) at 0° C. The mixture was allowed to warm to rt for 16 h, then quenched 34B. Ethyl with aqueous 2N HCl (3 mL) over 30 minutes. The mixture 2-(4-(4-hydroxyphenyl)cyclohexyl)acetate was partitioned between EtOAc and water and the layers were separated. The organic layers were washed with brine, To a solution ethyl 2-(4-(4-hydroxyphenyl)cyclohex 15 dried over sodium sulfate and concentrated under reduced ylidene)acetate (9.74 g., 35.8 mmol) in EtOAc was added pressure to afford a residue which was purified using silica Pd/C (0.974 g. 10 wt.%). The solution was sparged with a gel chromatography (15% EtOAc in hexanes) to deliver balloon of H (g) and stirred under an atmosphere of 1,1,1-trifluoro-3-(4-(4-methoxyphenyl)cyclohexyl)propan hydrogen for 2 days. The mixture was then filtered through 2-ol (565 mg, 71%) as a colorless oil. a pad of CELITE(R), which was thoroughly rinsed with EtOAc. The combined filtrate was then concentrated under 34G. 1,1,1-Trifluoro-3-(4-(4-methoxyphenyl)cyclo reduced pressure to afford ethyl 2-(4-(4-hydroxyphenyl) hexyl)propan-2-one cyclohexyl)acetate as a white crystalline Solid in quantitative yield as a mixture of diastereomers. To a solution of 1,1,1-trifluoro-3-(4-(4-methoxyphenyl) 25 cyclohexyl)propan-2-ol (34F, 565 mg, 1.89 mmol) in 34C. Ethyl CHCl (19 mL) was added sodium bicarbonate (476 mg, 2-(4-(4-methoxyphenyl)cyclohexyl)acetate 5.67) followed by Dess-Martin periodinane (1.04 g, 2.46 mmol) at 0°C. The mixture was stirred at rt for 16 h, then To a solution of ethyl 2-(4-(4-hydroxyphenyl)cyclohexyl) diluted with CHC1 and washed with water. The mixture acetate (34B, 34.1 g, 130 mmol) in DMF (300 mL) was 30 was concentrated under reduced pressure and the residue added CsCO, (65.0g, 200 mmol) followed by iodomethane was purified using silica gel chromatography (10% EtOAc (21.3 g 150 mmol). The resulting suspension was stirred at in hexanes) to afford 1,1,1-trifluoro-3-(4-(4-methoxyphenyl) rt for 16 h. The mixture was concentrated under reduced cyclohexyl)propan-2-one (356 mg, 63%) as a colorless oil pressure and the residue was partitioned between EtOAc which crystallized upon standing. (150 ml) and water (200 mL). The layers were separated and 35 the aqueous layer was extracted with EtOAc (3x150 mL). 34H. N-(4-Chlorophenyl)-1,1,1-trifluoro-3-(4-(4- These combined organic extracts were combined with the methoxyphenyl)cyclohexyl)propan-2-imine and (Z)- original organic layer and were dried over anhydrous 4-chloro-N-(3,3,3-trifluoro-1-(4-(4-methoxyphenyl) MgSO4, filtered, and concentrated under reduced pressure. cyclohexyl)prop-1-en-2-yl)aniline The residue was purified employing silica gel chromatog 40 raphy (0% to 30% EtOAc in hexanes) to afford ethyl A mixture of 1,1,1-trifluoro-3-(4-(4-methoxyphenyl)cy 2-(4-(4-methoxyphenyl)cyclohexyl)acetate as a clear oil. clohexyl)propan-2-one (34G, 178 mg, 0.59 mmol), 4-fluo roaniline (0.14 mL, 1.19 mmol) and p-TsOH (5 mg, 0.03 34D. 2-(4-(4-Methoxyphenyl)cyclohexyl)ethan-1-ol mmol), dissolved in toluene (5 mL), was heated to reflux 45 utilizing a Dean-Stark trap for 16 h. The mixture was Ethyl 2-(4-(4-methoxyphenyl)cyclohexyl)acetate (34C, concentrated under reduced pressure to afford a residue 1.45 g, 5.25 mmol) was dissolved in THF (26 mL) and which was purified by column chromatography on neutral cooled to 0°C. LiAlH4 (596 mg, 15.7 mmol) was then added alumina (7% EtOAc/hexanes) to give a mixture of imine portionwise, and the mixture was warmed to rt over 2 h. The N-(4-chlorophenyl)-1,1,1-trifluoro-3-(4-(4-methoxyphenyl) mixture was quenched with water, then 2N aqueous HCl, 50 cyclohexyl)propan-2-imine and (Z)-4-chloro-N-(3,3,3-trif before being extracted with EtOAc. The combined organic luoro-1-(4-(4-methoxyphenyl)cyclohexyl)prop-1-en-2-yl) phases were washed with brine, dried over sodium sulfate, aniline as a viscous, colorless oil. and concentrated under reduced pressure to furnish. 2-(4-(4- methoxyphenyl)cyclohexyl)ethan-1-ol (1.17 g, 95%). Example 34 55 34E. 4-Fluoro-N-(1,1,1-trifluoro-3-(4-(4-methoxyphenyl) 2-(4-(4-Methoxyphenyl)cyclohexyl)acetaldehyde cyclohexyl)propan-2-yl)aniline To a mixture of 2-(4-(4-methoxyphenyl)cyclohexyl) To a mixture of the product of 34H (N-(4-chlorophenyl)- ethan-1-ol (34D, 1.17 g. 5.00 mmol) in dichloromethane (50 60 1,1,1-trifluoro-3-(4-(4-methoxyphenyl)cyclohexyl)propan mL) was added sodium bicarbonate (1.26 g. 15.0 mmol), 2-imine and (Z)-4-chloro-N-(3,3,3-trifluoro-1-(4-(4- followed by Dess-Martin periodinane (3.19 g, 7.5 mmol) at methoxyphenyl)cyclohexyl)prop-1-en-2-yl)aniline) (34H, 0° C. The resultant mixture was stirred at rt for 16 h, before 160 mg, 0.41 mmol) in MeOH (10 mL), at rt, was added being diluted with dichloromethane then washed with water. sodium borohydride (46 mg, 1.2 mmol). The resultant The organic layer was concentrated under reduced pressure 65 mixture was stirred at rt for 1.5 h before being quenched to afford a residue which was adsorbed onto silica gel and with Satd. aq. ammonium chloride then extracted with purified using silica gel chromatography (20% EtOAC in dichloromethane. The combined organic phases were dried US 9,598.422 B2 77 78 over Sodium Sulfate and concentrated under reduced pres The following compound was made in the manner of sure. The resultant residue was purified by preparative General Procedure A, employing trans-4-phenyl-cyclo HPLC (Varian ProStar using Hamilton C18 PRP-1 column hexanamine (PCT Publication No. WO 2001/092204) (138 (15x250 mm) with flow rate of 20 mL/min, Mobile Phase A: mg, 0.79 mmol), 2-(4-cyanophenyl)acetic acid (138 mg, 0.5% formic acid in water; Mobile Phase B: 0.5% formic 0.86 mmol), 1-bis(dimethylamino)methylene)-1H-1,2,3- acid in acetonitrile; 0% to 100% B gradient elution during 30 minutes) to give a residue. The residue was diluted with 2M triazolo 4,5-b]pyridinium 3-oxid hexafluorophosphate (360 HCl in diethyl ether and concentrated under reduced pres mg, 0.95 mmol), and DMF (4 mL). The residue was purified sure to give the HCl salt of the desired compound as a to by preparative TLC (33% EtOAc in hexanes), followed by preparative HPLC (Varian ProStar using Hamilton C18 racemic mixture of cis/trans isomers. "H NMR (300 MHz: 10 CDC1): 8 7.16-7.08 (m, 2H), 6.95-6.81 (m, 4H), 6.65-6.59 PRP-1 column (15x250 mm) with flow rate of 20 mL/min, (m. 2H), 3.93-3.85 (m, 1H), 3.85-3.71 (m, 3H), 3.41 (d. Mobile Phase A: 0.5% formic acid in water; Mobile Phase J=9.0 Hz, 1H), 2.55-2.50 (m. 1H), 2.41 (tt, J=12.3, 3.0 Hz, B: 0.5% formic acid in acetonitrile; 0% to 100% B gradient 1H), 2.17-2.08 (m, 1H), 1.99-1.00 (m, 9H) ppm. m/z 396.15 elution during 30 minutes) to give the desired product. "H (M+H)". 15 NMR (300 MHz: CDC1): 8 7.64 (dd, J–8.4, 1.8 Hz, 2H), 7.40 (dd, J–8.4, 1.8 Hz, 2H), 7.32-7.24 (m, 2H), 7.21-7.16 Example 35 (m, 3H), 5.31 (d. J=7.5 Hz, 1H), 3.89-3.79 (m, 1H), 3.60 (s, 2H), 2.45 (tt, J=16.0, 3.6 Hz, 1H), 2.10-1.90 (m, 4H), 4-Chloro-N-(1,1,1-trifluoro-3-(4-(4-methoxyphenyl) 1.66-1.56 (m, 2H), 1.30-1.16 (m, 2H) ppm. m/z. 319 cyclohexyl)propan-2-yl)aniline hydrogenchloride (M+H)". Example 37 C 2-(4-Chlorophenyl)-N-((trans)-4-phenylcyclohexyl) 25 propanamide
NH HC 30 C O
HN H 35
Hill' Prepared utilizing the procedures used to afford 4-fluoro N-(1,1,1-trifluoro-3-(4-(4-methoxyphenyl)cyclohexyl)pro 40 pan-2-yl)aniline replacing 4-fluoroaniline with 4-chloroani line. MS(ES): m/z 412.10 M+H". to 2.94 min (Method M).
Example 36 45 2-(4-Cyanophenyl)-N-((trans)-4-phenylcyclohexyl) The following compound was made in the manner of acetamide General Procedure A, employing trans-4-phenyl-cyclo hexanamine (PCT Publication No. WO 2001/092204) (138 50 mg, 0.79 mmol), 2-(4-chlorophenyl)propanoic acid (158 mg, 0.86 mmol), 1-bis(dimethylamino)methylene)-1H-1.2. 2 3-triazolo 4,5-b]pyridinium 3-oxid hexafluorophosphate O (360 mg. 0.95 mmol), and DMF (4 mL). The residue was purified to by preparative TLC (33% EtOAc in hexanes) to 55 HN give a residue. The residue was further purified by prepara H tive HPLC (Varian ProStar using Hamilton C18. PRP-1 column (15x250 mm) with flow rate of 20 mL/min, Mobile Phase A: 0.5% formic acid in water; Mobile Phase B: 0.5% 60 formic acid in acetonitrile; 0% to 100% B gradient elution Hist' during 30 minutes) to give the desired product as a racemic mixture. "H NMR (300 MHz: CDC1): 8 7.34-7.14 (m, 9H), 5.16 (d. J–7.8 Hz, 1H), 3.86-3.75 (m. 1H), 3.48 (q, J–7.2 Hz, 65 1H), 2.42 (tt, J–12.3, 3.3 Hz, 1H), 2.09-1.84 (m, 4H), 1.68-1.52 (m, 2H), 1.49 (d. J–7.2 Hz, 3H), 1.26-1.06 (m, 2H) ppm. m/z 342 (M+H)". US 9,598.422 B2 79 80 Example 38 38D. 4-(Quinolin-4-yl)cyclohexan-1-amine
2-(4-Chlorophenyl)-N-((trans)-4-(duinolin-4-yl)cy The desired amine was made through reductive amination clohexyl)acetamide of 4-(quinolin-4-yl)cyclohexan-1-one with ammonium 5 acetate and Sodium cyanoborohydride to give 4-(quinolin 4-yl)cyclohexan-1-amine. C O 10 C O HN H NH2 HN H 15 Hist' Hill
r 2 21 N N Sa 2 N N 38A. 4-(1,4-Dioxaspiro4.5 dec-7-en-8-yl)guinoline 25 1,4-Dioxaspiro4.5dec-7-en-8-yl trifluoromethanesul 38E. 2-(4-Chlorophenyl)-N-((trans)-4-(duinolin-4- fonate (Bioorg. Med. Chem. Lett., 24:5377 (2014)) (6.9 g, yl)cyclohexyl)acetamide 23.9 mmol) was placed in a 500 mL round bottomed flask, followed by quinolone-4-boronic acid (4.55g, 26.3 mmol), 30 General Procedure A was employed using 4-(quinolin-4- Pd(PPh) (1.39 g, 1.2 mmol. 5 mol%), KBr (2.85g, 23.94 yl)cyclohexan-1-amine, and 2-(4-chlorophenyl)acetic acid. mmol) and sodium carbonate (6.34g, 59.85 mmol). The The residue was purified using preparative HPLC (Varian flask was evacuated and backfilled with N three times, ProStar using Hamilton C18 PRP-1 column (15x250 mm) before degassed dioxane (100 mL) and water (10 mL) were with flow rate of 20 mL/min, Mobile Phase A: 0.5% formic added to the solids and the mixture was stirred and heated to 35 90° C. under N, atmosphere. After 16 h, the mixture was acid in water, Mobile Phase B: 0.5% formic acid in acetoni cooled to rt, and SiO, was added. The mixture was concen trile; 0% to 100% B gradient elution during 30 minutes) to trated under reduced pressure and the residue was purified give the desired compound as a white powder. "H NMR (400 by silica gel column chromatography (0% to 100% EtOAc MHz: CDOD): 8 8.77-8.74 (m, 1H), 8.26-8.21 (m. 1H), in hexanes) to give 4-(1,4-dioxaspiro4.5 dec-7-en-8-yl)gui 40 8.05-8.01 (m, 1H), 7.79-7.73 (m. 1H), 7.68-7.63 (m, 1H), noline (3.2g, 50%). 7.49-7.45 (m. 1H), 7.34-7.27 (m, 4H), 3.82-3.77 (m, 1H), 3.50-3.42 (m, 3H), 2.13-2.03 (m, 4H), 1.82-1.70 (m, 2H), 38B. 4-(1,4-Dioxaspiro4.5 decan-8-yl)guinolone 1.65-1.60 (m, 2H) ppm. m/z. 379 (M+H)".
A mixture 4-(1,4-dioxaspiro4.5 dec-7-en-8-yl)guinolone 45 Example 40 (3.2 g, 12.0 mmol), NaHCO (500 mg. 6.0 mmol), and MeOH (70 mL) was purged with N, (g), before 20 wt.% of Pd/C (dry activated, 10 wt.%) was added to the mixture. He N—((R)-1-((1S,4S)-4-(6-Fluoroquinolin-4-yl)cyclo (g) was bubbled through the solution until complete disap hexyl)ethyl)-3-methylbenzenesulfonamide pearance of the starting material. The mixture was purged 50 with N (g), filtered through CELITE(R), and the filtrate was concentrated under reduced pressure. The residue was puri fied by flash chromatography to give 4-(1,4-dioxaspiro4.5
decan-8-yl)guinolone (2.9 g, 90%). 55 38C. 4-(Quinolin-4-yl)cyclohexan-1-one To a mixture of 4-(1,4-dioxaspiro4.5 decan-8-yl)gui nolone (3.0 g, 11 mmol) in acetone (30 mL) was added 3M aqueous HCl (30 mL). After the mixture was stirred at rt for 60 24 h, it was concentrated under reduced pressure and NaHCO (sat. aqueous solution) was added to adjust the pH above 8.0. The mixture was then extracted with EtOAc. The combined organic layers were dried with Na2SO4, and concentrated under reduced pressure to furnish 4-(quinolin 65 4-yl)cyclohexan-1-one as a yellow oil (2.3 g, 90%), which Solidified upon standing. US 9,598.422 B2 81 82 Preparation 40A: Preparation 40C:
O O
10 OTf
N N To a stirred solution of 1,4-dioxaspiro4.5 decan-8-one 15 o A mixture of Preparation 40B (368 g, 1.38 mol, 1.3 eq), (300 g, 1920.86 mmol. 1.0 eq) and phenyltrifluoromethane 4-Chloro-6-fluoroquinoline (195g, 1.07 mol. 1 eq), KCO, sulfonimide (823.47g, 2305.03 mmol, 1.2 eq) in MTBE (7.5 (445 g, 3.22 mol, 3 eq) and Pd(PPh) (25g, 22 mmol, 0.02 L) under N at -78°C. was added 2.0 M NaHMDS in THF eq) in dioxane-water (3 L., 4:1) was heated to reflux over night. The solution was then concentrated and extracted with (1152.2 mL, 2305.03 mmol. 1.2 eq) over 70 minutes, and the EtOAc. Purification by FCC (38% EtOAc/petroleum ether) mixture was stirred for an additional 60 minutes. The gave Preparation 40C (236 g, 77%). reaction mixture was warmed to room temperature and Preparation 40C: LC-MS. 286.1 (M+1)+, "H NMR (400 stirred overnight until TLC showed complete consumption MHz, CDC1) & 8.80-8.29 (d. 1H), 8.11-8.07 (q, 1H), of the starting material. The mixture was quenched with 7.63-7.61 (q, 1H), 7.47-7.46 (q, 1H), 7.26-7.22 (m. 1H), 25 5.75-5.74 (m. 1H), 408-405 (m, 4H), 2.63-2.59 (m, 2H), aqueous KHSO (100 ml), filtrated to remove the solid and 2.59-2.53 (m, 2H), 2.0-1.97 (m, 2H). concentrated the filtrate completely. To the residue was added 3 L MTBE, then washed with 5% NaOH (1.5 LX3). The organic phase was concentrated to obtain 567 g crude Preparation 40A (light yellow oil, yield 102%). The crude 30 can be used directly in next step without further purification. Preparation 40A: 'H NMR (400 MHz, CDC1): 8 (ppm) 5.65 (t, J=4.0 Hz, 1H), 3.98 (d. J=1.5 Hz, 4H), 2.53 (s. 2H), 2.40 (s. 2H), 1.90 (t, J=6.6 Hz, 2H) 35
N
40 Preparation 40D: To Preparation 40C (125.g., 0.44 mol) in IPA (2 L) at 55° C. was added 10% Pd/C and the mixture was stirred under an atmosphere of H overnight. The mixture was filtered and 45 concentrated to give crude Preparation 40D (130 g), which was used directly in the next step. 1 n
50
Preparation 40B: A mixture of crude Preparation 40A (600 g, 2.08 mol. 1 55 eq), BPin (687.1 g, 2.71 mol, 1.3 eq), KOAc (613 g, 6.24 21 mol. 3 eq), NaBr (86 g 0.833 mol, 0.4 eq) and Pd(dppf)Cl. N (76 g., 0.1 mol, 0.05 eq) in dioxane (6.5 L) was heated to reflux overnight. Once the reaction was complete, the mix 60 ture was concentrated and purified by FCC Preparation 40E: (2%->10%->20% EtOAc/PE) to give Preparation 40B (369 Preparation 40D (100 g, 0.348 mol) was treated with 4N HCl (300 mL) in acetone (1200 mL) at 45° C. overnight. The g, 66%). mixture was monitored by TLC. Then the solution was Preparation 40B: LC-MS: 267.1 (M+1)+, "H NMR (400 65 concentrated in vacuo. The residue was adjusted to pH 9 MHz, CDC1) & 6.46 (s, 1H), 3.98 (s, 4H), 2.37-2.35 (m, with 6 N NaOH. and the mixture was partitioned between 4H), 1.74-1.60 (t, 2H), 1.24 (s, 12H). ethyl acetate and water. The organic layer was washed with US 9,598.422 B2 83 84 brine, dried over anhydrous NaSO, filtered and concen To Preparation 40F (58.2 g 237.8 mmol) was added trated to give light yellow solid, which was then purified by MeCN (125 mL) and pyridine (38.7 mL, 480 mmol) and the silica gel column using hexanes and ethyl acetate (from 20 reaction mixture was cooled to 5°C. using an ice/water bath. percent ethyl acetate to 70% ethyl acetate) to afford Prepa Methanesulfonyl chloride (26.0 mL, 336 mmol) was added ration 40E as a white solid, (47 g--20 g mixture, yield >55%). Preparation 40E: LC-MS: 244.0 (M+1)+, "H NMR dropwise at 5°C. (exothermic reaction), the reaction mixture (400 MHz, CDC1) & 8.84 (d. J–4.6 Hz, 1H), 8.16 (dd, J=9.3, stirred for 1 hr at 5° C. and then brought up to room 5.7 Hz, 1H), 7.72 (dd, J=10.3, 2.8 Hz, 1H), 7.52 (ddd, J=9.2, temperature and stirred for an additional 16 h during which 7.8, 2.7 Hz, 1H), 7.29 (d. J=4.6 Hz, 1H), 3.69 (ddd, J=12.1, time a white precipitate formed. The heterogeneous mixture 9.0, 3.3 Hz, 1H), 2.77-2.54 (m, 4H), 2.37 (ddd, J=13.4, 5.9, 10 was quenched by the addition of Saturated aqueous ammo 3.0 Hz, 2H), 2.04 (qd, J=12.6, 5.3 Hz, 2H). nium chloride (200 mL) and extracted with CHCl (3x300 mL). The combined organic fractions were dried over anhy drous Sodium Sulfate and concentrated under reduced pres H 15 Sure. Excess pyridine was removed by azeotroping from Y toluene (3x300 mL). The crude material was recrystallized from H2O/MeOH as follows: 1 mL/mmol of HO was added and the slurry was heated to 120° C. in an oil bath. MeOH was added until the solids went into solution (-0.5 L). After cooling white crystals were collected by filtration to give 21 Preparation 40G (58.6 g. D20:1 dr, 76% over two steps). m/z. N (M+H)+=324.1. H-NMR (400 MHz: CDC1): 8 8.82 (dd. J–4.6, 0.2 Hz, 1H), 8.15-8.11 (m, 1H), 7.64-7.61 (m, 1H), 25 7.52-746 (m, 1H), 7.25 (s, 1H), 4.78 (tt, J=10.9, 5.2 Hz, Preparation 40F: 1H), 3.24-3.16 (m, 1H), 3.07 (d. J–1.0 Hz, 3H), 2.42-2.38 Preparation 40E (57.8 g. 237.8 mmol) was dissolved in (m. 2H), 2.16-2.12 (m. 2H), 1.93-1.66 (m, 4H). EtOH (240 mL) and cooled to 0° C. NaBH (9.94 g, 261.6 mmol) was added portionwise maintaining the temperature within a range of 0-10° C. (exothermic reaction). The 30 resulting suspension was stirred for 20 minutes. An LC/MS of an aliquot of the reaction mixture indicated consumption OH of ketone (m/z (M--H)+=244). The reaction was quenched at 0° C. by the slow addition of acetone (58 mL) over 15 35 minutes (exotherm). The reaction was poured slowly onto 500 mL of saturated aqueous ammonium chloride and 500 g of ice. The resulting aqueous Solution was extracted with EtOAc (3x300 mL) and the combined organic fractions were washed with Saturated aqueous ammonium chloride 40 21 (250 mL) and saturated aqueous sodium chloride (250 mL). The organic portion was dried over anhydrous sodium N sulfate and concentrated under reduced pressure. Sufficient silica to adsorb the oil was added and diluted with 10% MeOH in CHC1. A similar quantity of silica was used as 45 Preparation 40H: a silica plug to purify the material. The silica plug was Di-tert-butyl malonate (33.5 mL, 150 mmol) was added washed with 10% MeOH in CHC1 until UV-active mate dropwise to a stirred suspension of NaH (6.0 g. 60% rial no longer could be detected by TLC (7:3 EtOAc/ suspension in oil, 150 mmol) in 1,2-dimethoxyethane (100 Hexanes, R, 0.4). The filtrate was concentrated then sus mL) under Ar, cooled in a water-ice bath. After stirring for pended in 500 mL of toluene and concentrated again. Crude 50 10 min, Preparation 40G (16.2 g, 50 mmol) was added and Preparation 40F was isolated as a yellow solid (58.2 g) that the reaction was heated at 85°C. for 20 h. After this time, was used in the Subsequent step without further purification. acetic acid (100 mL) was added, the reaction flask was fitted Preparation 40G: with a distillation head and the temperature was raised to 130° C. 1,2-dimethoxyethane was distilled off under atmo 55 spheric pressure until the distillate was acidic (~100 mL). OMS The distillation head was removed, a reflux condenser was attached, water (20 mL) was added and the reaction heated at 130° C. for 12 h. The reaction was concentrated under reduced pressure and poured onto 200 g of ice and 100 mL 60 of saturated aqueous NaOAc. Preparation 40H was isolated as a white solid by filtration and further dried by refluxing with toluene in a Dean-Stark apparatus (11.0 g, 76%). m/z. 21 (M+H)"=288.2. 'H-NMR (400 MHz: DMSO-d): & 12.05 N (bs, 1H), 8.79 (d. J–4.5 Hz, 1H), 8.06 (dd, J=9.2, 5.8 Hz, 65 1H), 7.94 (dd, J=11.0, 2.8 Hz, 1H), 7.66-7.61 (m. 1H), 7.50 (d. J–4.6 Hz, 1H), 2.41 (d. J=7.6 Hz, 2H), 2.28-2.23 (m, 1H), 1.87-1.78 (m, 2H), 1.73-1.64 (m, 6H). US 9,598.422 B2 85 86 C. and LC/MS indicated the complete consumption of the starting material and formation of the desired methyl imide. The reaction mixture was rapidly diluted with saturated aqueous ammonium chloride solution (400 mL) and the biphasic mixture was stirred for 15 min. PrOAc (100 mL) y was added, the layers were separated, and the aqueous layer Ph was extracted with PrOAc (3x50 mL). The combined organic extracts were dried over anhydrous magnesium sulfate filtered, and concentrated. The resulting residue was 10 recrystallized by dissolving in 400 mL hot acetone and 21 adding HO until a milky solution formed followed to re-dissolving with heating (-3:1 acetone/HO). Preparation N 40J was obtained as white needles (15.04 g, 2 crops, 68%). 15 m/z (M+H)"=447.3. "H-NMR (400 MHz: CDC1): 88.81 (d. J=4.6 Hz, 1H), 8.10 (dd, J=9.2, 5.7 Hz, 1H), 7.65 (dd, Preparation 40I: J=10.6, 2.7 Hz, 1H), 7.47-7.42 (m. 1H), 7.41-7.29 (m, 6H), To a solution of Preparation 40H (1.4g, 4.8 mmol) in THF 5.47 (dd, J=8.8, 3.8 Hz, 1H), 4.69 (t, J=8.9 Hz, 1H), (15 mL) was added NEt (1.3 mL, 9.6 mmol). The reaction 4.38-4.30 (m, 1H), 4.26 (dd, J=8.9, 3.9 HZ, 1H), 3.26-3.21 mixture was cooled to 0° C. and trimethylacetyl chloride (m. 1H), 2.18-2.15 (m, 1H), 1.93-1.64 (m, 8H), 1.09 (d. (0.713 mL, 5.8 mmol) was added dropwise and the resulting J=6.9 Hz, 3H). solution stirred for 30 min at 0° C. In a separate flask, (R)-4-phenyloxazolidin-2-one (3, 1.01 g, 6.24 mmol) in THF (45 mL) at 0° C. was treated with 1 M LiHMDS solution in THF (dropwise addition of 6.24 mL, 6.24 mmol) 25 and stirred at 0°C. The lithiate was added via cannula to the first flask. The reaction mixture was allowed to warm to rt and was stirred for 3 hours. LC/MS indicated the complete consumption of the starting carboxylic acid and formation of the desired imide. The reaction mixture was poured onto 30 saturated aqueous ammonium chloride (50 mL) and the layers were separated. The aqueous layer was extracted with EtOAc (3x50 mL). The combined organic extracts were dried over anhydrous Sodium sulfate and chromatographed on silica using EtOAc/Hexanes 0 to 100% gradient to give 35 Preparation 40I as a white foam in 83% yield. m/z. (M--H)" =433.3. "H-NMR (400 MHz: CDC1): 8 8.80 (d. J=4.5 Hz, 1H), 8.11 (dd, J=9.1, 5.7 Hz, 1H), 7.63 (dd, J=10.5, 2.5 Hz, 1H), 7.48-7.43 (m. 1H), 7.40-7.30 (m, 6H), 5.47-5.44 (m, Preparation 40K: 1H), 4.71 (t, J=8.9 HZ, 1H), 4.31-4.28 (m, 1H), 3.20-3.11 (m, 40 To a solution of Preparation 40J (82.0 g, 183.6 mmol) in 3H), 2.49-2.46 (m. 1H), 1.82-1.67 (m, 6H). THF (610 mL) at 0°C. was added aqueous HO, (35 wt %, 82 mL) and LiOH (7.04 g, 293.8 mmol) in HO (189 mL).
The resulting reaction mixture was allowed to slowly warm to rt and stirred overnight. The reaction was cooled to 0°C. 45 and saturated aqueous sodium bisulfite solution (250 mL) was added. After stirring for 30 min, the THF was removed under reduced pressure. Acetic acid (34 mL) was added followed by EtOAc (300 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3x500 50 mL). The combined organic extracts were dried over anhy drous NaSO, filtered, and concentrated under reduced pressure. The brown crude reaction mixture was suspended in Mecn (400 mL) and the suspension was brought to reflux with vigorous stirring. After cooling to rt, the Solids were 55 collected by filtration washing with additional Mecn. Preparation 40K was obtained as a white solid (45.4 g. Preparation 40J: 82%). m/z (M+H)"=302.2. H-NMR (400 MHz: DMSO A solution of Preparation 40I (21.6 g. 50 mmol) in d6): 8 12.10 (s, 1H), 8.79 (d. J=4.5 Hz, 1H), 8.07 (dd, J=9.2, anhydrous THF (200 mL) was cooled to -40° C. (using 60 5.9 Hz, 1H), 7.97-7.94 (m, 1H), 7.67-7.62 (m. 1H), 7.49 (d. acetonitrile/dry ice bath, Some precipitation occurs) and 2 M J–4.5 Hz, 1H), 3.41-3.36 (m, 1H), 2.73-2.65 (m. 1H), NaHMDS solution in THF (30 mL, 60 mmol) was added 1.83-1.61 (m, 9H), 1.08 (d. J=6.8 Hz, 3H). Chiral HPLC, over 5 min (a 5-8°C. rise in temperature was observed). The >99% ee (ChiralPak IC-3, 3 uM, 4.6x250 mm, 15 min resulting yellow reaction mixture was stirred for 10 min, isocratic 70% heptane 30% i-ProH with 230 nm detection) became homogeneous, and Mel (10.6 g., 75 mmol) was 65 at a flow rate of 0.75 mL/min the desired enantiomer had a added dropwise over 2 min (a 10°C. rise in temperature was retention time of 8.6 min with the undesired enantiomer observed). The reaction mixture was stirred for 1 h at -40° eluting at 9.5 min. US 9,598.422 B2 88 Preparation 40L: sulfonyl chloride (26 mg, 0.147 mmol) and DCM (0.2 mL) followed directly after with the addition of DIPEA (64.1 ul, 0.367 mmol). The reaction was stirred at room temperature overnight. After overnight, the reaction was concentrated in 5 vacuo, taken up in 2 mL DMF, filtered, and purified via HPLC to give Example 40 (12.2 mg, 0.28 mmol. 39%) LC-MS Anal. Calc’d for CH,FNOS 426.18. found M+H 427.3 T=2.065 min (Method B). H NMR (500 MHz, DMSO-d) 8: 8.82 (d. J–4.5 Hz, 1H), 8.08 (dd, J=9.2, 10 5.8 Hz, 1H), 7.90 (dd, J=10.9, 2.5 Hz, 1H), 7.60-7.72 (m, 3H), 7.50 (d. J=8.5 Hz, 1H), 7.45 (t, J=7.7 Hz, 1H), 7.38 (d. J=7.6 Hz, 1H), 7.32 (d. J=4.5 Hz, 1H), 3.29 (t, J=10.5 Hz, 1H), 2.35 (s, 3H), 1.64-1.83 (m, 3H), 1.41-1.64 (m, 4H), Preparation 40K (2 g. 6.64 mmol) was taken up in toluene 1.23-1.41 (m, 2H), 0.92 (d. J=6.4 Hz, 3H). (22.12 ml) and diphenyl phosphorazidate (2.009 g, 7.30 15 mmol) and triethylamine (1.110 ml, 7.96 mmol) were added. Vial sealed and heated to 70° C. After 2 hours, the reaction was cooled to room temperature and concentrated under Examples 41 to 46 reduced pressure. Crude residue was taken up in 40 mL THF and 40 mL of water and lithium hydroxide (1.589 g, 66.4 mmol) was added. The reaction was stirred at room tem perature for 1 hour. The reaction was acidified with 1 NHCl (white precipitate forms) and extracted with EtOAc. The aqueous portion was then basified with 1N NaOH (precipi tate forms) and extracted with EtOAc 5 times. Basic extracts were concentrated in vacuo to give 40L (1.68 g. 6.17 mmol. 25 93% yield). LC-MS Anal. Calc’d for CHFN 272.17. found M+H 273.1 T-0.50 min (Method A). "H NMR (400 Mhz, chloroform-d) 8: 8.80 (d. J–4.6 Hz, 1H), 8.11 (dd. J=9.3, 5.7 Hz, 1H), 7.67 (dd, J=10.6, 2.8 Hz, 1H), 7.46 (ddd, J=9.2, 8.0, 2.8 Hz, 1H), 7.32 (d. J=4.5 Hz, 1H), 3.27-3.37 30 (m. 1H), 3.13 (dq, J=9.3, 6.3 Hz, 1H), 2.01-2.10 (m, 1H), 1.67-1.92 (m, 6H), 1.37-1.55 (m, 4H), 1.15 (d. J=6.4 Hz, 3H). Example 40 35 N—((R)-1-((1s.4S)-4-(6-Fluoroquinolin-4-yl)cyclo hexyl)ethyl)-3-methylbenzenesulfonamide Examples 41 to 46 were prepared from Preparation 40L Preparation 40L (20 mg, 0.073 mmol) was dissolved following the procedure for Example 40 using the corre DCM (0.2 mL) and added to a vial containing phenyl sponding Sulfonyl chlorides.
Tr Ex. No. Name R (min) Method B) M + H+
fluoroquinolin-4-yl)cyclohexyl) ethyl)-4-methylbenzenesulfonamide
42 2-chloro-N-((R)-1-((1s.4S)-4-(6- 2.085 447.2 fluoroquinolin-4-yl)cyclohexyl) ethyl)benzenesulfonamide
C
43 4-chloro-N-((R)-1-((1s.4S)-4-(6- C 2.126 447.2 fluoroquinolin-4-yl)cyclohexyl) ethyl)benzenesulfonamide
US 9,598.422 B2 95 96 Example 70 Intermediate 71A. Ethyl 2-(1,4-dioxaspiro4.5 decan-8-ylidene)acetate Triethyl phosphonoacetate (21.79 ml, 109 mmol) was added to a suspension of sodium hydride (3.84g, 96 mmol) in THF (64.0 ml) and 0° C. Reaction was stirred at room temperature for 30 minutes. After 30 minutes, the reaction was recooled to 0°C. and a solution of 1,4-dioxaspiro4.5 decan-8-one (10g, 64.0 mmol) in 5 mL THF was added. The 10 reaction was then stirred at room temperature for 30 minutes prior to quenching with water. The mixture was extracted with DCM three times. Combined organic extracts were dried with sodium sulfate, filtered, and concentrated in vacuo. Crude residue was purified via silica gel chromatog 15 raphy to give Intermediate 71A (13.88 g. 61.3 mmol. 96% yield). TLC: product stains as purple spot in anisaldehyde (Rf=0.75 in 1:1 Hex/EtOAc). H NMR (400 MHz, chloro form-d) 8: 5.65 (s, 1H), 4.13 (q, J=7.2 Hz, 2H), 3.92-3.99 (m, 4H), 2.94-3.02 (m, 2H), 2.31-2.40 (m, 2H), 1.71-1.79 (m, 4H), 1.26 (t, J–7.2 Hz, 3H). Intermediate 71B. Ethyl 2-(1,4-dioxaspiro4.5 decan-8-yl)acetate Example 70 25 Intermediate 71A (13.88 g. 61.3 mmol) was taken up in N—((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclo EtOAc (61.3 ml) and was added to a Parr hydrogenation hexyl)ethyl)-1,1'-biphenyl]-4-carboxamide bottle containing wet 10% palladium on carbon (1.306 g. 12.27 mmol) (54% w/w water) under an atmosphere of Preparation 40L (50 mg, 0.184 mmol) was taken up in nitrogen. The reaction bottle was purged and back-filled DMF (1836 ul) and HOBT (36.5 mg 0.239 mmol), EDC 30 with nitrogen three times, and then with hydrogen. After (45.8 mg, 0.239 mmol), 1,1'-biphenyl]-4-carboxylic acid filling the bottle with hydrogen to 50 psi, the bottle was (54.6 mg, 0.275 mmol) and TEA (128 ul, 0.918 mmol) were placed in a Parr shaker and shaken. After 4 hours, the added and reaction stirred at room temperature for 3 hours. reaction was filtered over pressed CELITER) and concen Reaction diluted with EtOAc and washed with 5:1 water/ trated in vacuo to give Intermediate 71B (13.78 g. 60.4 aqueous saturated NaHCO Solution. The combined organic 35 extracts were dried with sodium sulfate, filtered and con mmol, 98% yield). LC-MS Anal. Calc’d for CHO centrated in vacuo. The crude residue was purified via silica 228.14. found M+H 229.1 T, 0.83 min (Method A). H gel flash column chromatography to give Example 70 (63 NMR (400 MHz, chloroform-d) 8: 4.11 (q, J–7.2 Hz, 2H), mg, 0.132 mmol, 72.0% yield). LC-MS Anal. Calc’d for 3.88-3.95 (m, 4H), 2.2.1 (d. J–7.0 Hz, 2H), 1.83 (dqd, CHFNO, 452.23. found M+H 453.3 T=2.297 min J=11.0, 7.5, 3.5 Hz, 1H), 1.68-1.78 (m, 4H), 1.50-1.61 (m, (Method B). H NMR (400 MHz, CHLOROFORM-d) 8: 40 2H), 1.27-1.35 (m, 2H), 1.24 (t, J–7.2 Hz, 3H). 8.82 (d. J–4.5 Hz, 1H), 8.13 (dd, J=9.2, 5.7 Hz, 1H), 7.85 (d. J=8.6 Hz, 2H), 7.64-7.70 (m, 3H), 7.58-7.64 (m, J=70 Hz, Intermediate 71C.. Ethyl 2H), 7.36-7.50 (m, 5H), 5.91 (d. J=9.2 Hz, 1H), 4.58-470 2-(1,4-dioxaspiro4.5 decan-8-yl)butanoate (m. 1H), 3.30 (tt, J=10.6, 3.6 Hz, 1H), 1.96-2.15 (m, 3H), 45 Diisopropylamine (2.347 ml, 16.63 mmol) taken up in dry 1.80 (br. s. 6H), 1.33 (d. J=6.6 Hz, 3H). THF (15.99 ml) (under N atmosphere) and cooled to -78° Example 71 C. n-Bulli (6.14 ml, 15.35 mmol) (2.5 M in hexanes) was added over ~5 minutes at -78° C. After stirring for 45 4-Chloro-N-(1-((trans)-4-(duinolin-4-yloxy)cyclo minutes, reaction was warmed to room temperature for 10 hexyl)propyl)benzamide 50 minutes and returned to -78°C. Then, 1,3-dimethyltetrahy dropyrimidin-2(1H)-one (1.541 ml, 12.79 mmol) was added followed by a solution of Intermediate 71B (2.92 g, 12.79 C mmol) in THF (15.99 ml) (dropwise over-5 minutes). After
1 hour, iodoethane (1.125 ml, 14.07 mmol) (neat) was added dropwise over-5 minutes. Reaction stirred another 2 hours 55 at -78°C. before slowly warming to room temperature. The reaction was then stirred over night at room temperature. The reaction was quenched by pouring into 1:1 water/brine and extracting with EtOAc. Combined organics washed with brine, dried with sodium sulfate, filtered and concentrated in 60 vacuo. Crude residue was purified via silica gel column chromatography to give Intermediate 71C (2.27 g, 8.86 mmol. 69% yield). TLC: product stains as purple spot in anisaldehyde (Rf 0.80 in 1:1 hex/EtOAc). H NMR (400 MHz, chloroform-d) 8: 4.14 (q, J–7.5 Hz, 2H), 3.88-3.95 (m, 65 4H), 2.09 (td, J=8.4, 5.6 Hz, 1H), 1.69-1.83 (m, 4H), 1.45-1.64 (m, 6H), 1.33-1.42 (m, 1H), 1.25 (t, J=7.1 Hz, 3H), 0.86 (t, J–7.5 Hz, 3H). US 9,598.422 B2 97 98 Intermediate 71D. Ethyl Intermediate 71H. 1-((trans)-4-(Quinolin-4-yloxy) 2-(4-oxocyclohexyl)butanoate cyclohexyl)propan-1-amine Intermediate 71C (2.00 g, 7.80 mmol) was taken up in Intermediate 71G (190 mg, 0.606 mmol) was taken up in 5 toluene (2021 ul) in a vial and diphenyl phosphorazidate THF (39.0 ml) and hydrochloric acid, 1M (39.0 ml) was (184 mg. 0.667 mmol) and TEA (101 ul, 0.728 mmol) were added. Reaction stirred at room temperature for 2 hours. The added. The vial sealed and heated to 80° C. After 2 h, the reaction was concentrated in vacuo, diluted with water and reaction was cooled to room temperature and concentrated extracted with EtOAc. The combined organic extracts were under reduced pressure. The crude residue was taken up in dried with sodium sulfate, filtered and concentrated in 1 mL THF and 1 mL of water and LiOH (145 mg, 6.06 vacuo. The crude material was purified on silica gel column 10 mmol) was added. Reaction stirred at room temperature for chromatography to give Intermediate 71 D (1.47 g. 6.92 1 hour. The reaction was acidified with 1N HCl (white mmol. 89% yield). TLC: product stains faintly pink in precipitate forms) and extracted with EtOAc to remove anisaldehyde (Rf 0.65 in 1:1 Hex/EtOAc). "H NMR (400 DPPA related impurities. Then, the reaction was basified MHz, chloroform-d) 8: 4.15 (q, J=7.1 Hz, 2H), 2.25-2.42 (m, with 1N NaOH (precipitate forms again) and extracted with 4H), 2.18 (ddd, J=9.3, 7.8, 5.2 Hz, 1H), 2.10 (ddt, J=13.1, 15 EtOAc (x5). Basic extracts were concentrated in vacuo to 6.2, 3.3 Hz, 1H), 1.90-2.03 (m, 2H), 1.56-1.70 (m, 2H), give Intermediate 71H (35 mg, 0.123 mmol. 20.30% yield). 1.38-1.56 (m, 2H), 1.25 (t, J=7.2 Hz, 3H), 0.89 (t, J–74 Hz, LC-MS Anal. Calc'd for C.H.N.O. 284.19. found M+H 3H). 285.2 T-0.55 min (Method A). Intermediate 71 E. Ethyl Example 71 2-((trans)-4-hydroxycyclohexyl)butanoate 4-Chloro-N-(1-((trans)-4-(duinolin-4-yloxy)cyclo Intermediate 71 D (1.47 g. 6.92 mmol) was dissolved in hexyl)propyl)benzamide EtOH (13.85 ml) and cooled to 0° C. NaBH (0.314.g., 8.31 mmol) was added and the reaction was then allowed to stir 25 Intermediate 71H (35 mg, 0.123 mmol) was taken up in at 0°C. for 1 hour. The reaction was quenched with saturated DMF (1231 ul) and HOBT (24.50 mg, 0.160 mmol), EDC aqueous NHCl and extracted with EtOAc. Combined (30.7 mg, 0.160 mmol), 4-chlorobenzoic acid (38.5 mg. organic extracts were dried with sodium Sulfate, filtered, and 0.246 mmol) and TEA (86 ul, 0.615 mmol) were added and concentrated in vacuo. The crude material was purified via reaction was stirred at rt. After 2 hours, the reaction was silica gel column chromatography to give Intermediate 71E 30 diluted with DMF filtered and purified via preparative HPLC (1.22 g, 5.69 mmol. 82% yield) along with (138 mg 0.644 to give Example 71 (24.6 mg, 0.058, 46.8% yield). LC-MS mmol, 9.30% yield) of the cis-isomer. "H NMR (400 MHz, Anal. Calcd for CHCINO, 422.18. found M+H 423.3 chloroform-d) 8: 4.14 (q, J=7.1 Hz, 2H), 3.53 (t, J=10.5 Hz, T-0.82 min (Method A). "H NMR (500 MHz, DMSO-d) 1H), 1.92-2.08 (m, 2H), 1.80-1.89 (m. 1H), 1.63-1.70 (m, 8: 8.67 (d. J=5.2 Hz, 1H), 8.15 (d. J=9.0 Hz, 1H), 8.11 (d. 1H), 1.52-1.62 (m, 4H), 1.37-1.52 (m, 2H), 1.26 (t, J=7.2 35 J=8.2 Hz, 1H), 7.87-7.94 (m, 3H), 7.72 (t, J=7.6 Hz, 1H), Hz, 3H), 0.95-1.17 (m, 2H), 0.87 (t, J=74 Hz, 3H). 7.49-7.58 (m, 3H), 7.10 (d. J=5.2 Hz, 1H), 4.62 (t, J=10.2 HZ, 1H), 3.74-3.85 (m, J=8.9 HZ, 1H), 2.21 (d. J–10.1 Hz, Intermediate 71F. Ethyl 2-((trans)-4-(duinolin-4- 2H), 1.86 (t, J=14.3 Hz, 2H), 1.39-1.71 (m, 5H), 1.28 (q, yloxy)cyclohexyl)butanoate J=12.3 Hz, 2H), 0.85 (t, J–7.2 Hz, 3H). 40 Intermediate 71E (100 mg, 0.467 mmol) was taken up in Enantiomer 1 and Enantiomer 2 DMSO (933 ul) and NaH (22.40 mg, 0.933 mmol) was added slowly, portionwise at rt. After 1 hour, 4-bromoqui Enantiomer 1 noline (117 mg, 0.560 mmol) was added and the reaction was heated to 80° C. After 16 hours, the reaction was 45 Example 71 quenched with ammonium chloride and extracted with EtOAc. The combined organic extracts were dried with 4-Chloro-N-(1-((trans)-4-(duinolin-4-yloxy)cyclo sodium sulfate, filtered, concentrated in vacuo. The crude hexyl)propyl)benzamide (Homochiral, Stereochem residue was purified via silica gel column chromatography istry Unknown) to give Intermediate 71 F (89 mg, 0.261 mmol, 55.9% yield). 50 LC-MS Anal. Calc’d for CH, NO. 341.20. found M+H 342.3T, 0.84 min (Method A).
Intermediate 71G. 2-((trans)-4-(Quinolin-4-yloxy) cyclohexyl)butanoic acid 55 Intermediate 71 F (89 mg 0.261 mmol) was taken up in THF (1043 ul), Water (1043 ul), and MeOH (521 ul). Lithium hydroxide (62.4 mg, 2.61 mmol) was added and reaction stirred at 60° C. for 24 hours. The reaction was 60 concentrated in vacuo, diluted with water, acidified with acetic acid added (precipitate forms), and extracted with EtOAc. The combined organic extracts were dried with Sodium Sulfate, filtered and concentrated in vacuo to give Intermediate 71G (74 mg., 0.236 mmol, 91% yield. LC-MS 65 Anal. Calc’d for CHNO 313.17. found M+H 314.2 T=0.69 min (Method A).
US 9,598.422 B2 101 102 tography to give Intermediate 73B (369 g, 66% yield). (-2:1 ratio). LC-MS Anal. Calc’d for CHFN 254.12. LC-MS Anal. Calc’d for CHBO 266.17. found M+H found M+H 255.1, rt=0.60 (first elution diastereomer) and 267.1. "H NMR (400 MHz, CDC1) & 6.46 (s, 1H), 3.98 (s, 0.62 min (second eluting diastereomer) (Method A). 4H), 2.37-2.35 (m, 4H), 1.74-1.60 (t, 2H), 1.24 (s, 12H). Intermediate 73G. Intermediate 73C. 6-Fluoro-4-(1,4-dioxaspiro4.5 4-(6-Fluoroquinolin-4-yl)cyclohexanecarboxylic dec-7-en-8-yl)cquinoline acid A mixture of Intermediate 73B (368 g, 1.38 mol, 1.3 eq), Intermediate 73F (280 mg, 1.101 mmol) taken up in 4-chloro-6-fluoroquinoline (195g, 1.07 mol. 1 eq), KCO 10 methanol (5505 ul) and hydrochloric acid, 37% (5505 ul). (445 g, 3.22 mol, 3 eq) and Pd(PPh) (25g, 22 mmol, 0.02 Reaction heated at 70° C. After 48 hours, the reaction was eq) in dioxane-water (3 L., 4:1) was heated to reflux over slowly added to 100 mL water and basified with sodium night. The solution was concentrated and extracted with bicarbonate (sat aq). The aqueous was extracted with EtOAc. The crude residue was purified via silica gel column EtOAc. The combined organics were dried with sodium chromatography to give Intermediate 73C (236 g, 77% 15 Sulfate, filtered, and concentrated in vacuo to give crude yield). LC-MS Anal. Calc’d for CHFNO. 285.12. found methyl 4-(6-fluoroquinolin-4-yl)cyclohexanecarboxylate. M+H 286.1. "H NMR (400 MHz, CDC1) & 8.80-8.29 (d. This crude material was taken up in THF (4260 ul), Water 1H), 8.11-8.07 (q, 1H), 7.63-7.61 (q, 1H), 7.47-7.46 (q, 1H), (4260 ul), MeOH (2130 ul) and lithium hydroxide (255 mg. 7.26-7.22 (m. 1H), 5.75-5.74 (m, 1H), 408-4.05 (m, 4H), 10.65 mmol) was added. Reaction stirred at room tempera 2.63-2.59 (m, 2H), 2.59-2.53 (m, 2H), 2.0-1.97 (m, 2H). ture for 1 hour. The reaction was then concentrated, acidified with 1N HC1 and extracted with EtOAC. The combined Intermediate 73D. 6-Fluoro-4-(1,4-dioxaspiro4.5 organics were dried with sodium sulfate, filtered, and con decan-8-yl)guinoline centrated in vacuo to give a crude residue. This crude residue was purified via silica gel column chromatography to give To Intermediate 73C (125 g, 0.44 mol) in IPA (2 L) at 55° 25 Intermediate 73G (mixture of cis and trans) (63 mg, 0.231 C. was added 10% Pd/C and the mixture was stirred under mmol. 21.64% yield). LC-MS Anal. Calc’d for an atmosphere of H overnight. The mixture was filtered and CHFNO. 273.1. found M+H 274.1, rt=0.55 (Method concentrated to give crude Intermediate 73D (128 g. 100% A). yield), which was used directly in the next step. LC-MS Anal. Calc’d for C.H.FNO. 287.13. found M+H 288.2, 30 Example 73 and Example 74 rt=0.62 min (Method A). N-(4-Chlorobenzyl)-4-(6-fluoroquinolin-4-yl)cyclo Intermediate 73E. hexanecarboxamide (Mixture of Cis- and Trans 4-(6-Fluoroquinolin-4-yl)cyclohexanone Isomers) 35 Intermediate 73D (100 g, 0.348 mol) was treated with 4 Intermediate 73G (15 mg 0.055 mmol) was dissolved in N HCl (300 mL) in acetone (1200 mL) at 45° C. for thionyl chloride (40.1 ul, 0.549 mmol) and DMF (2.125ul, overnight. Then the solution was concentrated in vacuo. The 0.027 mmol) was added. Reaction stirred at room tempera residue was adjusted pH 9 with 6 N NaOH. The mixture was ture for 1 hour. After, the reaction was concentrated in partitioned between ethyl acetate and water. The organic 40 vacuo, taken up in toluene, concentrated again and placed on layer was washed with brine, dried over anhydrous NaSO, high vac. After 15 minutes, the crude acyl chloride was taken filtered and concentrated to give light yellow solid, which up in ACN (274 ul) and added to a solution of (4-chloro was then purified by silica gel column chromatography to phenyl)methanamine (15.54 mg., 0.110 mmol) in ACN (274 afford Intermediate 73E as white solid (67 g, 55% yield). ul) and TEA (38.2 ul, 0.274 mmol) at 0° C. Reaction was LC-MS Anal. Calc’d for CHFNO, 243.11. found M+H 45 then allowed to warm to room temperature. After 1 hour, the 244.0. "H NMR (400 MHz, CDC1) & 8.84 (d. J=4.6 Hz, reaction was diluted with water and extracted with EtOAc. 1H), 8.16 (dd, J=9.3, 5.7 Hz, 1H), 7.72 (dd, J=10.3, 2.8 Hz, Organics were dried with sodium sulfate, filtered, and con 1H), 7.52 (ddd, J=9.2, 7.8, 2.7 Hz, 1H), 7.29 (d. J=4.6 Hz, centrated in vacuo. Crude residue taking up in DMF filtered, 1H), 3.69 (ddd, J=12.1, 9.0, 3.3 Hz, 1H), 2.77-2.54 (m, 4H), and purified via preparative HPLC to give two diastereom 2.37 (ddd, J=13.4, 5.9, 3.0 Hz, 2H), 2.04 (qd, J=12.6, 5.3 Hz, 50 CS. 2H). The first eluting diastereomer. Example 73 (4.9 mg, 0.012 mmol, 22% yield). LC-MS Anal. Calc’d for CHCIFNO, Intermediate 73F. 396.14. found M+H 397.0, rt=1.891 (Method B). H NMR 4-(6-Fluoroquinolin-4-yl)cyclohexanecarbonitrile (500 MHz, DMSO-d) 8: 8.80 (d. J–4.4 Hz, 1H), 8.40 (t, 55 J=5.7 Hz, 1H), 8.08 (dd, J=9.0, 5.9 Hz, 1H), 7.99 (d. J=9.8 To a solution of Intermediate 73E (500 mg, 2.055 mmol) HZ, 1H), 7.66 (t, J=8.6 Hz, 1H), 7.45 (d. J=4.3 Hz, 1H), 7.37 and 1-((isocyanomethyl)sulfonyl)-4-methylbenzene (522 (d. J=8.2 Hz, 2H), 7.26 (d. J–8.1 Hz, 2H), 4.26 (d. J=5.8 Hz, mg, 2.67 mmol) in DMSO (10.100 ml) and methanol (0.202 2H), 3.33 (t, J=11.9 Hz, 1H), 2.32 (t, J=11.9 Hz, 1H), 1.92 ml) was added potassium 2-methylpropan-2-olate (554 mg. (t, J=11.2 Hz, 4H), 1.71-1.83 (m, 2H), 1.56 (q, J=12.3 Hz, 4.93 mmol). After the addition was complete, the reaction 60 2H). mixture was stirred room temperature. After 1 hour, the The second eluting enantiomer, Example 74 (3.6 mg, reaction was diluted with diethyl ether (30 mL) and washed 0.009 mmol, 17% yield) LC-MS Anal. Calc’d for with water (20 ml). The organic layer was dried with CHCIFNO, 396.14. found M+H 397.0, rt=1.940 anhydrous MgSO4, concentrated under reduced pressure. (Method B). H NMR (500 MHz, DMSO-d) 8: 8.78 (d. The crude material was purified via silica gel column 65 J=4.3 Hz, 1H), 8.37 (t, J=5.5 Hz, 1H), 8.07 (dd, J=8.9, 6.0 chromatography to give Intermediate 73F (280 mg, 1.101 HZ, 1H), 7.95 (d. J=10.8 Hz, 1H), 7.65 (t, J=8.6 Hz, 1H), mmol. 53.6% yield) as a mixture of cis and trans isomers 7.36 (d. J=8.0 Hz, 2H), 7.23-7.31 (m,3H), 4.27 (d. J=5.7 Hz, US 9,598.422 B2 103 104 2H), 3.33 (br. S., 1H), 2.61 (br. S., 1H), 2.01-2.13 (m, 2H), Intermediate 77A. Ethyl 2-((cis)-4-(pyridin-4-yloxy) 1.74-1.85 (m, 4H), 1.65-1.74 (m, 2H). cyclohexyl)butanoate Example 75 and Example 76 Intermediate 71E (100 mg, 0.467 mmol) was dissolved in (trans)-N-(4-Chlorophenyl)-4-(6-fluoroquinolin-4- THF (1867 ul) and pyridin-4-ol (98 mg, 1.027 mmol) and yl)cyclohexanecarboxamide (cis)-N-(4-Chlorophe triphenylphosphine (269 mg, 1.027 mmol) were added. nyl)-4-(6-fluoroquinolin-4-yl)cyclohexanecarboxam Solution was cooled to 0° C. in an ice bath. Diisopropyl aZodicarboxylate (200 ul, 1.027 mmol) was added and the ide (Homochiral, Absolute and Relative reaction was allowed to stir at room temperature once the Stereochemistry Unassigned) addition was complete. Stirred at room temperature for 16 10 hours. Reaction was concentrated in vacuo and purified via silica gel column chromatography to give Intermediate 77A (89 mg, 0.205 mmol, 43.9% yield). LC-MS Anal. Calc’d for CHNO. 291.18. found M+H 292.3 T, 0.84 min (Method A). "H NMR (400 MHz, chloroform-d) 8: 8.34 15 8.42 (m, 2H), 6.71-6.79 (m, 2H), 4.57-4.64 (m, 1H), 4.15 (q, C C J=7.1 Hz, 2H), 2.14 (ddd, J=9.8, 7.9, 4.6 Hz, 1H), 1.97-2.07 (m. 2H), 1.38-1.69 (m, 9H), 1.24-1.29 (m,3H), 0.88 (t, J–7.4 Hz, 3H) Intermediate 77B. 2-((cis)-4-(Pyridin-4-yloxy)cyclo N hexyl)butanoic acid 2 Intermediate 77A (89 mg, 0.305 mmol) was taken up in N THF (244 ul), water (244 ul), and MeOH (122 ul). Lithium 25 hydroxide (73.1 mg, 3.05 mmol) was added and the reaction Example 75 and Example 76 were made from Interme stirred at 60° C. for 16 hours. Lithium hydroxide (73.1 mg, diate 73G utilizing the procedure to make Example 73 and 3.05 mmol) was again added and the reaction stirred for Example 74. another 24 hours at 60° C. The reaction was concentrated in First eluting diastereomer: (7.1 mg, 0.018 mmol. 34% vacuo, diluted with water and extracted with EtOAc. The yield) LC-MS Anal. Calc’d for CHCIFNO, 382.13. 30 aqueous layer was then treated with AcOH and extracted found M+H 383.2, rt=2.011 (Method B). H NMR (500 with EtOAc. LCMS shows Product remains in aqueous MHz, DMSO-d) 8: 10.00 (s, 1H), 8.78 (d. J=44 Hz, 1H), layer. Extracted again with 7:3 chloroform: propanol. LCMS 8.07 (dd, J=9.1, 5.8 Hz, 1H), 7.96 (d. J=8.8 Hz, 1H), Shows product was successfully extracted from the aqueous 7.59-7.68 (m, 3H), 7.39 (d. J=44 Hz, 1H), 7.33 (d. J=8.8 Hz, layer. The combined organic layers were dried with sodium 2H), 3.37 (br. S., 1H), 2.78 (br. S., 1H), 2.09 (d. J=12.1 Hz, 35 2H), 1.81-2.00 (m, 4H), 1.75 (d. J=11.4 Hz, 2H). Sulfate, filtered, and concentrated in vacuo to give Interme Second eluting diastereomer: (8.4 mg. 0.021 mmol. 39% diate 77B (73 mg, 0.277 mmol, 91% yield). Material was not yield) LC-MS Anal. Calc’d for CHCIFNO, 382.13. further purified. LC-MS Anal. Calc’d for CHNO found M+H 383.0, rt=1.988 (Method B). H NMR (500 263.15. found M+H 264.2 T=0.58 min (Method A). MHz, DMSO-d) 8: 10.09 (s, 1H), 8.81 (d. J–4.4 Hz, 1H), 8.08 (dd, J=9.0, 5.9 Hz, 1H), 8.01 (d. J=8.9 Hz, 1H), 40 Intermediate 77C. 1-((cis)-4-(Pyridin-4-yloxy)cyclo 7.6.1-7.71 (m, 3H), 7.46 (d. J=44 Hz, 1H), 7.34 (d. J=8.7 Hz, hexyl)propan-1-amine 2H), 3.36 (t, J=11.9 HZ, 1H), 2.41-2.48 (m, 1H) (triplet buried under DMSO), 1.97 (d. J=9.7 Hz, 4H), 1.76-1.88 (m, Intermediate 77B (35 mg, 0.133 mmol) was taken up in 2H), 1.54-1.65 (m, 2H). toluene (443 jul) and diphenyl phosphoraZidate (40.2 mg, 45 0.146 mmol) and TEA (22.23 ul, 0.159 mmol) added. The Example 77a reaction vial was sealed (vented with needle while reaching temperature) and heated to 80°C. After 2 h, the reaction was (+)-4-Chloro-N-(1-((cis)-4-(pyridin-4-yloxy)cyclo cooled to room temperature and concentrated under reduced hexyl)propyl)benzamide pressure. The crude residue was taken up in 1 mL THF and 50 1 mL of water LiOH (31.8 mg, 1.329 mmol) was added. The reaction stirred at room temperature overnight. The reaction was acidified with 1N HCl and extracted with EtOAc C (extracts discarded). The aqueous portion was then basified with 1N NaOH and extracted with EtOAc (x2). The com 55 bined basic, organic extracts were dried with sodium sulfate, filtered and concentrated in vacuo to give Intermediate 77C (25 mg, 0.107 mmol. 80% yield). LC-MS Anal. Calc’d for CHNO, 234.17. found M+H 235.1 T=0.43 min (Method A). 60 Example 77a (+)-4-Chloro-N-(1-((cis)-4-(pyridin-4-yloxy)cyclo hexyl)propyl)benzamide 65 Intermediate 77C (25 mg, 0.107 mmol) was taken up in DMF (1067 ul) and HOBT (21.24 mg., 0.139 mmol), EDC
US 9,598.422 B2 109 110 81 A. tert-Butyl 4-(4-chlorobenzamido)methyl)-4- Example 84 hydroxypiperidine-1-carboxylate N-(1,1'-Biphenyl-4-yl)-4-(6-fluoroquinolin-4-yl) piperazine-1-carboxamide A solution of tert-butyl 4-(aminomethyl)-4-hydroxypip eridine-1-carboxylate (0.25 g, 1.086 mmol) and 4-chlo robenzoic acid (0.204 g, 1.303 mmol) in DMF (2 mL) was treated with triethylamine (0.454 mL, 3.26 mmol) followed by BOP (0.576g, 1.303 mmol). The reaction was stirred for 10 2 h then quenched with dil. aq. HOAc. This resulted in the formation of a precipitate, so the mixture was filtered and rinsed with water. It was then Suspended in dil. aq. Sodium bicarbonate, Sonicated, then filtered, rinsed with water, and 15 air-dried to afford tert-butyl 4-(4-chlorobenzamido) methyl)-4-hydroxypiperidine-1-carboxylate (0.38 g. 90% yield) as a colorless solid, mp 172-173° C. MS(ES): s NH m/z. 369 M+H". t-O.93 min (Method A). ON 81B. 4-Chloro-N-((4-hydroxypiperidin-4-yl)methyl) benzamide, HCl 25
A solution of HCl (3.56 ml, 14.23 mmol) in dioxane was treated with tert-butyl 4-(4-chlorobenzamido)methyl)-4- 30 hydroxypiperidine-1-carboxylate (0.35 g, 0.949 mmol). Ini tially, material dissolved as it was added, but eventually, a 84A. tert-Butyl gum formed. This was stirred for 15 min. then ~3 mL of 4-(6-fluoroquinolin-4-yl)piperazine-1-carboxylate dichloromethane was added and stirring was continued for 2 h. During this time, product took on the form of a finely 35 To a homogeneous mixture of 4-chloro-6-fluoroquinoline (500.0 mg, 2.8 mmol) in NMP (5 mL), in a sealable vial, was divided Suspension. Concentration under reduced pressure added 1-Boc-piperazine (750.0 mg. 4.0 mmol) followed by afforded 4-chloro-N-(4-hydroxypiperidin-4-yl)methyl)ben DIPEA (2 mL, 11.6 mmol). After the addition was complete, Zamide, HCl (0.29 g, quantitative yield) as a white powder. the vial was capped and the mixture was stirred at 120° C. MS(ES): m/z 269 M+H". t-O.55 min (Method A). 40 After 15 hours, the reaction was cooled to room temperature then partitioned between water and EtO. The layers were separated and the aqueous layer was extracted twice more Example 81 with EtO then once with EtOAc. These organic extracts were combined with the original organic layer and were 4-Chloro-N-((1-(6-fluoroquinolin-4-yl)-4-hydroxypi 45 washed with brine, dried (NaSO), filtered and concen peridin-4-yl)methyl)benzamide trated in vacuo to afford the crude product as an oil. Purification by Isco chromatography afforded tert-butyl 4-(6-fluoroquinolin-4-yl)piperazine-1-carboxylate as a solid A Suspension of 4-chloro-6-fluoroquinoline (0.119 g, (719.3 mg: 77% yield). MS(ES): m/z 332 M+H". t—0.70 0.655 mmol) and 4-chloro-N-((4-hydroxypiperidin-4-yl) 50 min (MethodA). "H NMR (400 MHz, DMSO-d) & 8.70 (d. methyl)benzamide, HCl (0.2g, 0.655 mmol) in NMP (1 mL) J-49 Hz, 1H), 8.04 (dd, J=9.2, 5.6 Hz, 1H), 7.76-7.58 (m, was treated with DIEA (0.286 mL, 1.638 mmol) and heated 2H), 7.07 (d. J=5.0 Hz, 1H), 3.71-3.54 (m, 4H), 3.14-3.01 to 135° C. for 5 h. After about 45 min. the reaction had (m, 4H), 1.44 (s, 9H). become homogeneous. The reaction was cooled to ~80° C. and treated with ~3 mL of 5% aq. HOAc resulting in the 55 formation of a precipitate. This was stirred briefly, filtered, 84B. 6-Fluoro-4-(piperazin-1-yl)cquinoline rinsed several times with water and once with 10% EtOAc hexanes, and air-dried to afford 4-chloro-N-(1-(6-fluoro To a homogeneous mixture of tert-butyl 4-(6-fluoroqui quinolin-4-yl)-4-hydroxypiperidin-4-yl)methyl)benzamide nolin-4-yl)piperazine-1-carboxylate (700.0 mg, 2.1 mmol) (0.21 g, 74% yield) as an off-white solid, mp 91-94° C. 60 in anhydrous dioxane (4 mL), at room temperature under MS(ES): m/z 414 M+H". to 0.68 min (Method A). "H nitrogen, was added HCl (4N in dioxane, 10 mL, 40.0 NMR (400 MHz, DMSO-d) & 8.67 (d. 1H, J=4.9 Hz), 8.46 mmol). After 6 hours, a precipitate had formed which was (t, 1H, J=6.1 Hz), 7.99-8.04 (m, 1H), 7.94 (d. 2H, J=8.7 Hz), isolated by vacuum filtration, rinsed with anhydrous dioxane 7.55-7.63 (m, 4H), 7.05 (d. 1H, J=5.0 Hz), 4.71 (s, 1H), 3.42 and dried under vacuum to afford the title compound (508.0 (d. 2H, J=6.1 Hz), 3.24-3.31 (m, integration obscured by 65 mg, 79% yield) as an HCl salt which was used without water peak), 3.10-3.18 (m, 2H), 1.85-1.94 (m, 2H), 1.67 further purification. MS(ES): m/Z 232 M+H". to 0.38 1.72 (m, 2H). min (Method A). US 9,598.422 B2 111 112 84 N-(1,1'-Biphenyl-4-yl)-4-(6-fluoroquinolin-4- 86B. (1-(6-Fluoroquinolin-4-yl)piperidin-4-yl)meth yl)piperazine-1-carboxamide anamine
To a heterogeneous mixture of the HCl salt of 6-fluoro To a homogeneous mixture of tert-butyl (1-(6-fluoroqui 4-(piperazin-1-yl)guinoline (84B, 25.0 mg, 0.09 mmol) in 5 nolin-4-yl)piperidin-4-yl)methyl)carbamate (308.1 mg, 0.9 anhydrous DMF (1 mL), at room temperature, was added mmol) in DCM (10 mL), under nitrogen atmosphere, was DIPEA (0.05 mL, 0.29 mmol) followed by 4-isocyanato-1, added TFA (1.2 mL, 15.6 mmol). The resultant mixture was 1'-biphenyl (23.0 mg, 0.12 mmol). The resulting mixture stirred at ambient temperature for 45 minutes before being was stirred for 96 hours, before being diluted with DMF, concentrated in vacuo to afford the TFA salt of the title passed through a syringe filter, then purified via preparative 10 compound as a gold oil, which was used without further HPLC/MS to afford the title compound (12.9 mg: 21% purification. MS(ES): m/z. 260 M+H". to 0.43 min yield). MS(ES): m/z 427 M+H". t-1.55 min (Method I). (Method A). H NMR (500 MHz, DMSO-d) & 8.69 (d. J=6.5 Hz, 1H), Example 86 8.07 (dd, J=9.2, 5.1 Hz, 1H), 7.99-7.96 (m, 1H), 7.93-7.90 15 (m. 1H), 7.63 (d. J=7.7 Hz, 2H), 7.58-7.57 (m, 4H), 7.44 7.41 (m, 2H), 7.33-7.26 (m, 2H), 7.20 (d. J=6.6 Hz, 1H), N-((1-(6-Fluoroquinolin-4-yl)piperidin-4-yl)methyl)- 3.87-3.81 (m, 4H), 3.81-3.73 (m, 4H). 4-methylbenzamide Example 86 To a homogeneous mixture of the TFA salt of (1-(6- fluoroquinolin-4-yl)piperidin-4-yl)methanamine (86B, 41.8 N-((1-(6-Fluoroquinolin-4-yl)piperidin-4-yl)methyl)- 4-methylbenzamide mg, 0.09 mmol), 4-methylbenzoic acid (14.0 mg 0.1 mmol) 25 and DIPEA (0.06 mL, 0.3 mmol) in anhydrous THF (1 mL), dioxane (0.5 mL) and DMF (0.5 mL), under nitrogen atmosphere, was added PyBOP (44.6 mg, 0.09 mmol). After stirring at ambient temperature for 15 hours, the mixture was diluted with DMSO, passed through a syringe filter, then 30 purified via preparative HPLC/MS to afford the title com pound (21.1 mg: 65% yield). MS(ES): m/z. 378 M+H". t=1.25 min (Method I). H NMR (500 MHz, DMSO-d) & 8.63-8.53 (m, 2H), 7.97 (dd, J=8.9, 5.7 Hz, 1H), 7.70 (d. 35 J=7.9 Hz, 2H), 7.63-7.51 (m, 2H), 7.25 (d. J=7.8 Hz, 2H), 7.00 (d. J=4.8 Hz, 1H), 3.47 (d. J=11.4 Hz, 2H), 2.76 (t, J=11.6 Hz, 2H), -2.53 (m, integration, exact chemical shift range obscured by solvent peak), 2.31 (s.3H), 1.87-1.73 (m. 21 3H), 1.55-1.41 (m, 2H). 40 N Example 87 3,4-Dichloro-N-((1-(6-fluoroquinolin-4-yl)piperidin 4-yl)methyl)benzamide 86A. tert-Butyl (1-(6-fluoroquinolin-4-yl)piperidin 45 4-yl)methyl)carbamate
C To a homogeneous mixture of 4-chloro-6-fluoroquinoline (220.0 mg, 1.2 mmol) in anhydrous NMP (4 mL), in a 50 sealable vial, was added tert-butyl (piperidin-4-ylmethyl) C carbamate (350.0 mg, 1.6 mmol) followed by DIPEA (0.8 mL, 4.6 mmol). The vial was sealed and the mixture was stirred at 60° C. for 2 hours, then at 90° C. for 17 hours before being stirred at 120° C. for 24 hours. After cooling to 55 room temperature, the reaction mixture was purified by Isco silica gel chromatography to afford tert-butyl (1-(6-fluoro 21 quinolin-4-yl)piperidin-4-yl)methyl)carbamate as an off white solid (323.7 mg: 74% yield). MS(ES): m/z 360 60 S. M+H". t-0.71 min (Method A). "H NMR (400 MHz, N DMSO-d) & 8.66 (d. J=5.0 Hz, 1H), 8.01 (dd, J=9.1, 5.7 Hz, To a heterogeneous mixture of the TFA salt of (1-(6- 1H), 7.66-7.51 (m, 2H), 7.01 (d. J=4.9 Hz, 1H), 6.93 (t, fluoroquinolin-4-yl)piperidin-4-yl)methanamine (86B, 41.8 J=5.7 Hz, 1H), 3.48 (d. J=12.2 Hz, 2H), 2.94 (t, J=6.3 Hz, 65 mg, 0.09 mmol) in anhydrous THF (1 mL) and dioxane (0.5 2H), 2.76 (t, J=11.2 Hz, 2H), 1.80 (d. J=11.1 Hz, 2H), mL), under nitrogen atmosphere, was added DIPEA (0.06 1.67-1.55 (m, 1H), 1.51-1.42 (m, 2H), 1.40 (s, 9H). mL, 0.3 mmol) followed by 3,4-dichlorobenzoyl chloride US 9,598.422 B2 113 114 (18.9 mg, 0.09 mmol). After stirring at ambient temperature Example 91 for 16 hours, the mixture was diluted with DMF, filtered through a syringe filter, then purified via preparative HPLC/ 1-(1-(6-Fluoroquinolin-4-yl)piperidin-4-yl)-3-(p- MS to afford the title compound (23.1 mg; 62% yield). tolyl)urea MS(ES): m/z. 432 M+H". t1.51 min (Method I). "H NMR (500 MHz, DMSO-d) & 8.83-8.76 (m, 1H), 8.63 (d. J=4.8 Hz, 1H), 8.07 (s, 1H), 7.99 (dd, J=8.8, 5.7 Hz, 1H), 7.82 (d. J=8.2 Hz, 1H), 7.73 (d. J=8.4 Hz, 1H), 7.64-7.54 (m, 2H), 7.01 (d. J=4.8 Hz, 1H), 3.71-3.44 (m, 2H), 3.34-3.21 HN (m. 2H), 2.76 (t, J=11.7 Hz, 2H), 1.88-1.75 (m, 3H), 10 1.56-145 (m, 2H). ins Examples 88 to 90 15 Reaction of the TFA salt of (1-(6-fluoroquinolin-4-yl) piperidin-4-yl)methanamine with an appropriate acid chlo N ride, under the conditions described for Example 87 F (Scheme 1, below), afforded compounds of the invention shown in Table 1 below. N CCN Scheme 1 91A. tert-Butyl (1-(6-fluoroquinolin-4-yl)piperidin 25 4-yl)carbamate To a homogeneous mixture of 4-chloro-6-fluoroquinoline (200.0 mg, 1.1 mmol) in anhydrous NMP (5 mL), in a sealable vial, was added 4-Boc-aminopiperidine (309.0 mg. 30 1.5 mmol) followed by DIPEA (0.8 mL, 4.6 mmol). The vial was sealed and the mixture was stirred at 120° C. for 15 hours. After cooling to room temperature, the reaction mixture was partitioned between EtOAc and water. The layers were separated and the aqueous layer was extracted 35 twice more with EtOAc. The organic extracts were com bined, washed with brine, dried (NaSO), filtered and concentrated in vacuo to afford the crude product which was used without further purification, based on quantitative yield. MS(ES): m/z 346 M+H". to 0.70 min (Method A). TABLE 1. 40 91B. 1-(6-Fluoroquinolin-4-yl)piperidin-4-amine Ex. tR To a homogeneous mixture of tert-butyl (1-(6-fluoroqui No. R (M + H)" (min.y.ethod nolin-4-yl)piperidin-4-yl)carbamate (380.0 mg, 1.1 mmol) 45 in dioxane (2 mL), under nitrogen atmosphere, was added 4M HCl in dioxane (2 mL, 8.0 mmol). The resultant mixture was stirred at ambient temperature for 6 hours, during which time a precipitate formed. Vacuum filtration afforded the HCl salt of the title compound as a pale yellow solid (358 50 mg, 100% yield) which was used without further purifica tion. MS(ES): m/z 246 M+H". to.42 min (Method A). Example 91 1-(1-(6-Fluoroquinolin-4-yl)piperidin-4-yl)-3-(p- 55 tolyl)urea To a heterogeneous mixture of the HCl salt of 1-(6- fluoroquinolin-4-yl)piperidin-4-amine (91B, 30.0 mg 0.09 mmol) in anhydrous THF (1 mL), at room temperature, was 90 398 1.33 60 added DIPEA (0.05 mL, 0.29 mmol) followed by 1-isocya nato-4-methylbenzene (15.1 mg, 0.11 mmol). The resulting mixture was stirred for 88 hours, before being diluted with DMSO, passed through a syringe filter, then purified via preparative HPLC/MS to afford the title compound (21.7 xO. 65 mg: 60% yield). MS(ES): m/z. 379 M+H". t1.30 min (Method I). H NMR (500 MHz, DMSO-d) & 8.67 (d. J–2.5 HZ, 1H), 8.29 (s, 1H), 8.11-7.96 (m, 1H), 7.70-7.57 (m, 2H),
US 9,598.422 B2 117 118 119A. 5-lum particles; Mobile Phase A: 5:95 acetonitrile: water trans-4-(6-Fluoroquinolin-4-yl)cyclohexanamine with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradi To a solution of 4-(6-fluoroquinolin-4-yl)cyclohexanone ent: 30-70% B over 22 minutes, then a 5-minute hold at (350 mg, 1.439 mmol) in EtOH (6 mL) in a microwave vial 100% B; Flow: 20 mL/min. Fractions containing the desired was added ammonium acetate (1663 mg, 21.58 mmol). The product were combined and dried via centrifugal evapora resulting Suspension was treated with sodium cyanoborohy tion. The yield of the product was 11.4 mg (0.029 mmol. dride (108 mg, 1.726 mmol). The reaction was capped and 67%) "H NMR (400 MHz, DMSO-d) & 8.82 (d. J–4.5 Hz, microwaved at 130° C. for 5 min. The reaction was cooled 1H), 8.50 (s, 1H), 8.10 (dd, J=9.3, 5.9 Hz, 1H), 8.03 (dd, to RT and diluted with MeOH and purified by preparative 10 J=11.1, 2.8 Hz, 1H), 7.68 (td, J=8.7, 2.8 Hz, 1H), 7.50 (d. HPLC (PHENOMENEX(R) Luna 5u 30x100 mm), 40 J=4.5 Hz, 1H), 7.46-7.35 (m, 2H), 7.33-7.19 (m, 2H), 6.19 mL/min flow rate with gradient of 0% B-100% B over 12 (d. J–7.7 Hz, 1H), 3.70-3.52 (m. 1H), 3.42-3.34 (m. 1H), minutes. Hold at 100% B for 2 min. (A: 0.1% TFA in 2.04 (d. J=9.3 Hz, 2H), 1.92 (d. J=12.1 Hz, 2H), 1.79-1.63 water/MeOH (90:10), B: 0.1% TFA in water/MeOH (10:90) (m. 2H), 1.61-1.45 (m, 2H) MS: Anal. Calc’d for monitoring at 254 nm. Fractions containing the product were 15 CHCIFNO, 397.136. found M+H 398.2 LC: t-1.39 combined and concentrated to give trans-4-(6-fluoroquino min (Method I). lin-4-yl)cyclohexanamine (310 mg. 0.624 mmol. 43.4% These compounds were obtained following the proce yield) H NMR (400 MHz, DMSO-d) & 8.88 (d. J=4.6 Hz, dures in Example 119 using the corresponding isocyanates. 1H), 8.19-8.05 (m, 2H), 7.92 (br. s. 3H), 7.73 (td, J=8.7, 2.8 HZ, 1H), 7.53 (d. J=4.6 Hz, 1H), 3.36 (br. s. 1H), 3.22-3.02 (m. 1H), 2.09 (br. S., 2H), 1.96 (br. S., 2H), 1.77-1.52 (m, 4H) O MS: Anal. Calc’d for CHFN 244.138. found M+H 245.1 LC: t-O.42 min (Method A) HN ls N1 R E H Example 119 25 1-(4-Chlorophenyl)-3-(trans-4-(6-fluoroquinolin-4- yl)cyclohexyl)urea
To a solution of trans-4-(6-fluoroquinolin-4-yl)cyclo 30 F hexanamine (20 mg, 0.042 mmol) in THF (0.5 mL) at RT N was added 1-chloro-4-isocyanatobenzene (9.76 mg, 0.064 2 mmol). The reaction was stirred at RT for 3 h. The crude N material was purified via preparative LC/MS with the fol lowing conditions: Column: XBridge C18, 19x200 mm,
Ex. Tr No. Name R (min) Method M + H" 120 1-(4-chloro-2-fluorophenyl)-3-(trans-4- F 1.46 416.O (6-fluoroquinolin-4-yl)cyclohexyl)urea / 121 1-(4-cyanophenyl)-3-trans-4-(6- fluoroquinolin-4-yl)cyclohexyl)urea 7. 1.16 389.1 CN
122 1-(2,4-difluorophenyl)-3-(trans-4-(6- F 1.25 399.9 fluoroquinolin-4-yl)cyclohexyl)urea / 1231-(3,4-difluorophenyl)-3-(trans-4-(6- 1.37 400.2 fluoroquinolin-4-yl)cyclohexyl)urea /or US 9,598.422 B2 119 120 -continued
Ex. Tr No. Name R (min) Method 1 M + H+. 1241-(4-fluorophenyl)-3-(trans-4-(6- 1.26 382.3 fluoroquinolin-4-yl)cyclohexyl)urea
Example 125 added 1-chloro-4-isocyanatobenzene (16.27 mg, 0.106 mmol). The reaction was stirred at RT for 2 h. The crude 1-(4-Chlorophenyl)-3-(cis-4-(6-fluoroquinolin-4-yl) 15 material was purified via preparative LC/MS with the fol cyclohexyl)urea lowing conditions: Column: XBridge C18, 19x200 mm, 5-lum particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradi C ent: 30-70% B over 22 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evapora ls tion. The yield of the product was 14.7 mg (0.034 mmol. 64%) 'H NMR (500 MHz, DMSO-d) & 8.95 (d. J=4.5 Hz, 25 1H), 8.56 (s, 1H), 8.21-8.08 (m, 2H), 7.78 (t, J=8.5 Hz, 1H), 7.63 (d. J=44 Hz, 1H), 7.43 (d. J=8.3 Hz, 2H), 7.27 (d. J=7.4 Hz, 2H), 6.69 (d. J–7.4 Hz, 1H), 4.01 (br. S., 1H), 3.59 (d. J=9.4 Hz, 1H), 1.96-1.78 (m, 4H), 1.76 (br. S., 4H) MS: 30 Anal. Calc'd for CHCIFNO, 397.136. found M+H N 398.2 LC: tr—1.44 min (Method I).
2 Example 130 trans-N-Benzyl-4-(6-fluoroquinolin-4-yl)cyclo 35 hexanamine 125A. cis-4-(6-Fluoroquinolin-4-yl)cyclohexanamine
To a solution of 4-(6-fluoroquinolin-4-yl)cyclohexanone 40 H (350 mg, 1.439 mmol) in EtOH (6 mL) in a microwave vial was added ammonium acetate (1663 mg, 21.58 mmol). To the resulting Suspension was added sodium cyanoborohy dride (108 mg, 1.726 mmol). The reaction was capped and microwaved at 130° C. for 5 min. The reaction was cooled 45 to RT and diluted with MeOH and purified by preparative HPLC (PHENOMENEX(R) Luna 5u 30x100 mm), 40 N mL/min flow rate with gradient of 0% B-100% B over 12 minutes Hold at 100% B for 2 min. (A: 0.1% TFA in 2 water/MeOH (90:10), B: 0.1% TFA in water/MeOH (10:90) 50 N monitoring at 254 nm. Fractions containing the product were combined and concentrated to give cis-4-(6-fluoroquinolin To a solution of 4-(6-fluoroquinolin-4-yl)cyclohexanone 4-yl)cyclohexanamine (100 mg, 0.201 mmol, 14% yield) "H (100 mg, 0.411 mmol) and beny Zlamine (66 mg, 0.617 NMR (400 MHz, DMSO-d) & 8.94 (d. J–46 Hz, 1H), mmol) in CHCl (2 mL) at RT was added acetic acid (0.024 8.19-8.03 (m, 2H), 7.94 (br. S., 1H), 7.74 (td, J=8.7, 2.8 Hz, 55 mL, 0.411 mmol), followed by sodium triacetoxyborohy 1H), 7.60 (d. J=4.8 Hz, 1H), 3.59 (br. s., 1H), 3.49 (t, J=11.0 dride (131 mg, 0.617 mmol). The reaction was stirred at RT HZ, 1H), 2.10-182 (m, 6H), 1.75 (d. J=10.8 Hz, 2H) MS: for 4 h. Then it was diluted with MeOH and purified with Anal. Calc’d for CHFN 244.138. found M+H 245.1 prep HPLC (Phen Luna 5u 30x100 mm), 40 mL/min flow LC: t-0.45 min (Method A). rate with gradient of 0% B-100% B over 12 minutes Hold at 60 100% B for 2 min. (A: 0.1% TFA in water/MeOH (90:10), Example 125 B: 0.1%TFA in water/MeOH (10:90) monitoring at 254 nm. Evaporation of the product containing fractions gave (1r, 1-(4-Chlorophenyl)-3-(cis-4-(6-fluoroquinolin-4-yl) 4r)-N-benzyl-4-(6-fluoroquinolin-4-yl)cyclohexanamine cyclohexyl)urea (150 mg). An aliquot (15 mg) of this material was further 65 purified under the following conditions: Column: XBridge To a solution of cis-4-(6-fluoroquinolin-4-yl)cyclohexan C18, 19x200 mm. 5-lum particles; Mobile Phase A: 5:95 amine (25 mg, 0.053 mmol) in THF (0.5 mL) at RT was acetonitrile: water with 10-mMammonium acetate; Mobile
US 9,598.422 B2 123 124 (0.024 mL, 0.169 mmol). The reaction was stirred at RT for evaporation. The yield of the product was 11.5 mg (0.029 3 h. The crude material was purified via preparative LC/MS mmol. 68%) 'H NMR (500 MHz, DMSO-d) & 8.76 (d. with the following conditions: Column: XBridge C18, J=4.5 Hz, 1H), 8.25 (d. J–7.7 Hz, 1H), 8.07 (dd, J=9.1, 5.8 19x200 mm. 5-lum particles; Mobile Phase A: 5:95 acetoni HZ, 1H), 7.93 (d. J=8.8 Hz, 1H), 7.71-7.58 (m. 1H), 7.46 (d. trile: water with 10-mMammonium acetate; Mobile Phase J=44 Hz, 1H), 7.36-7.30 (m, J=8.2 Hz, 2H), 7.29-7.13 (m, B: 95:5 acetonitrile: water with 10-mMammonium acetate; J=8.2 Hz, 2H), 3.94-3.81 (m, 2H), 3.61 (d. J=7.3 Hz, 1H), Gradient: 50-100% B over 20 minutes, then a 5-minute hold 3.25 (t, J=11.2 Hz, 1H), 1.88 (t, J=13.7 Hz, 4H), 1.66-1.43 at 100% B; Flow: 20 mL/min. Fractions containing the (m, 4H) MS: Anal. Calc’d for CHCIFNO, 396.140. desired product were combined and dried via centrifugal found M+H 397.0 LC: t-1.37 min (Method I).
Ex. Tr No. Name R (min) Method 1 M + H+.
145 2-(4-chlorophenyl)-N-(trans-4- C 1.52 411.1 (6-fluoroquinolin-4- O yl)cyclohexyl)propanamide HN
N F
2 N
1464-chloro-N-(trans-4-(6- O 1.42 383.2 fluoroquinolin-4- yl)cyclohexyl)benzamide HN
C
F N
2 N
147 2-(4-chlorophenyl)-N-(cis-4-(6- 1.75 397.2 fluoroquinolin-4- yl)cyclohexyl)acetamide
US 9,598.422 B2 127 128 -continued 1M hydrogen chloride (aqueous) (0.929 mL, 3.71 mmol). C The mixture was heated at 50° C. for 6 h. The reaction
mixture was concentrated. The residue was dissolved in EtOAc, washed with water (2x), and brine. The solution was dried over NaSO and concentrated. The crude material was purified with ISCO (EtOAc/Hex 0-30%). Fractions contain ing product were concentrated to yield ethyl 2-(4-oxocyclo hexyl)propanoate (290 mg, 79% yield) as a clear oil. "H NMR (400 MHz, CHLOROFORM-d) & 4.22-4.06 (m, 2H), 10 2.46-2.30 (m, 5H), 2.13-1.91 (m, 3H), 1.56-142 (m, 2H), 1.31-1.24 (m, 3H), 1.18 (d. J=7.1 Hz, 3H). 157D. Ethyl 2-(4-(((trifluoromethyl)sulfonyl)oxy) cyclohex-3-en-1-yl)propanoate 15 C Ethyl 2-(4-oxocyclohexyl)propanoate (200 mg, 1.01 mmol) and 2,6-di-tert-butyl-4-methylpyridine (238 mg, 1.16 mmol) were dissolved in dry DCM (10 ml). To the reaction mixture trifluoromethanesulfonic anhydride (0.186 mL, 1.11 mmol) was added dropwise and stirred for 2 h. The suspen sion was filtered and the filtrate was diluted with DCM, washed with 1N HCl (2x), satd. sodium bicarbonate solu tion, water, and brine. The solution was dried over NaSO and concentrated to yield ethyl 2-(4-(((trifluoromethyl)sul 25 fonyl)oxy)cyclohex-3-en-1-yl)propanoate (320 mg. 96% yield) as a brown oil. "H NMR (400 MHZ, CHLORO FORM-d) & 5.73 (t, J=6.1 Hz, 1H), 428-4.05 (m, 2H), 2.52-2.17 (m, 4H), 2.08-1.79 (m, 3H), 1.49 (dt, J=11.1, 6.6 HZ, 1H), 1.31-1.20 (m, 3H), 1.19-1.04 (m, 3H) 30 157A. Ethyl 157E. Ethyl 2-(4-(4.4.5.5-tetramethyl-1,3,2-dioxa 2-(1,4-dioxaspiro4.5 decan-8-ylidene)propanoate borolan-2-yl)cyclohex-3-en-1-yl)propanoate To a suspension of NaH (0.307 g., 7.68 mmol) in THF (8 To a solution of ethyl 2-(4-(((trifluoromethyl)sulfonyl) mL) cooled at 0°C. was added ethyl 2-(diethoxyphosphoryl) 35 oxy)cyclohex-3-en-1-yl)propanoate (300 mg. 0.908 mmol) propanoate (1.830 g, 7.68 mmol) slowly. After 30 min, in DMSO (5 mL) was added 4.4.4.4.5.5.5',5'-octamethyl 1,4-dioxaspiro4.5 decan-8-one (1 g, 6.40 mmol) was 2,2'-bi(1,3,2-dioxaborolane) (230 mg. 0.908 mmol) and added. The resulting mixture was stirred at 0°C. for 2 h, then potassium acetate (267 mg, 2.72 mmol). After the mixture warmed to RT overnight. The mixture was quenched with was degassed with N for 10 minutes, PdCl(dppf) (19.9 mg, water and the THF was removed in vacuo. The residue was 40 0.027 mmol) was added. The mixture was heated at 80° C. dissolved in EtOAc, washed with water and brine. The overnight. The mixture was partitioned between EtOAc and solution was dried over NaSO, filtered and concentrated. water. The organic phase was concentrated and purified by The crude material was purified by ISCO (EtOAc/Hex ISCO silica gel column. Fractions containing product were 0-30%). Fractions containing the product were concentrated concentrated to yield ethyl 2-(4-(4,4,5,5-tetramethyl-1,3,2- to yield ethyl 2-(1,4-dioxaspiro4.5 decan-8-ylidene)pro 45 dioxaborolan-2-yl)cyclohex-3-en-1-yl)propanoate (168 mg, panoate (1.2g, 78% yield) a light yellow oil. "H NMR (400 60% yield) as a brown oil. "H NMR (400 MHz, CHLORO MHz, CHLOROFORM-d) & 4.19 (q, J=7.1 Hz, 2H), 4.03 FORM-d) & 6.66-6.40 (m, 1H), 4.31-4.00 (m, 2H), 2.34-2.26 3.89 (m, 4H), 2.68-2.53 (m, 2H), 2.46-2.28 (m, 2H), 1.89 (s. (m. 1H), 2.25-2.19 (m, 1H), 2.19-2.04 (m, 2H), 1.95-1.75 3H), 1.78-1.66 (m, 4H), 1.30 (t, J=7.1 Hz, 3H). (m, 3H), 1.73-1.60 (m, 1H), 1.29-1.24 (m, 15H), 1.13 (dd. 157B. Ethyl 50 2-(1,4-dioxaspiro4.5 decan-8-yl)propanoate J=11.6, 7.0 Hz, 3H) 157F. Ethyl 2-(4-(2-methylpyridin-4-yl)cyclohex-3- A suspension of ethyl 2-(1,4-dioxaspiro4.5 decan-8- en-1-yl)propanoate ylidene)propanoate (500 mg, 2.081 mmol) and 10% palla dium on carbon (25 mg, 0.024 mmol) in EtOAc (5 mL) was 55 To a solution of ethyl 2-(4-(4,4,5,5-tetramethyl-1,3,2- hydrogenated in a Parr shaker at 45 psi for 6 h. The catalyst dioxaborolan-2-yl)cyclohex-3-en-1-yl)propanoate (120 mg. was filtered and the filtrate was concentrated to yield ethyl 0.389 mmol) in dioxane (3 mL) was added 4-bromo-2- 2-(4-(3-methylpyridin-4-yl)cyclohexyl)propanoate (450 methylpyridine (67.0 mg 0.389 mmol), water (1 mL) and mg, 89% yield) as a light oil. "H NMR (400 MHZ, CHLO NaCO (165 mg, 1.557 mmol). The mixture was degassed ROFORM-d) & 4.12 (dtt, J=10.7, 7.1, 3.6 Hz, 2H), 3.98-3.81 60 with N2 for 10 minutes. Pd(PhP) (22.49 mg, 0.019 mmol) (m, 4H), 2.35-2.17 (m, 1H), 1.83-1.68 (m, 3H), 1.66-1.45 was then added. The mixture was heated to 100° C. for 16 (m, 4H), 1.43-1.28 (m, 2H), 1.27-1.22 (m, 3H), 1.14-1.07 h. The cooled mixture was diluted with EtOAc, washed with (m, 3H) water and brine. The solution was dried over NaSO, 157C. Ethyl 2-(4-oxocyclohexyl)propanoate filtered and evaporated. The crude material was purified by 65 ISCO silica gel chromatography (0-50% EtOAc/Hexane). To a solution of ethyl 2-(1,4-dioxaspiro4.5 decan-8-yl) Fractions containing product were concentrated to yield propanoate (450 mg, 1.857 mmol) in THF (5 mL) was added ethyl 2-(4-(2-methylpyridin-4-yl)cyclohex-3-en-1-yl)pro US 9,598.422 B2 129 130 panoate (100 mg, 0.366 mmol, 94% yield) as a yellow oil. (m, 4H), 1.50-1.11 (m, 3H), 1.08 (dd, J=6.4, 2.8 Hz, 3H). "H NMR (400 MHz, chloroform-d) & 8.61-8.11 (m. 1H), MS: Anal. Calc’d for CHN, 218.18. found M+H 219.2 7.09-6.68 (m, 2H), 4.15 (qdd, J=7.1, 3.3, 1.8 Hz, 2H), LC: t-O.43 min. 2.71-2.57 (m, 1H), 2.53 (d. J=5.3 Hz, 3H), 2.47-2.35 (m, Example 157a 0.5H), 2.29 (t, J=7.1 Hz, 0.5H), 1.98-1.75 (m, 3H), 1.67-1.38 (m, 4H), 1.32-1.22 (m, 4H), 1.21-1.09 (m, 4H). 4-Chloro-N-(1-(4-(2-methylpyridin-4-yl)cyclohexyl) ethyl)benzamide 157G Ethyl 2-(4-(2-methylpyridin-4-yl)cyclohexyl)propanoate 10 N Ethyl 2-(4-(2-methylpyridin-4-yl)cyclohex-3-en-1-yl) N propanoate (100 mg, 0.366 mmol) was dissolved in MeOH (5 mL). Ammonium formate (115 mg, 1.829 mmol) and 21 palladium on carbon (10%) (10.51 mg 0.099 mmol) were added. The vessel was equipped with a reflux condenser, 15 evacuated and flushed with N three times. The reaction was then heated to reflux. After one hour, the reaction was cooled and filtered. The filtrate was concentrated in vacuo. The residue was dissolved in EtOAc, washed with sodium bicar bonate solution, water, and brine. The solution was dried over NaSO filtered and concentrated. The crude product N was used directly in the next step. C 157H. 25 2-(4-(2-Methylpyridin-4-yl)cyclohexyl)propanoic To a solution of 1-(4-(2-methylpyridin-4-yl)cyclohexyl) acid ethanamine (100 mg 0.458 mmol) (157I) in THF (2 mL) was added 4-chlorobenzoic acid (108 mg 0.687 mmol), To a mixture of ethyl 2-(4-(2-methylpyridin-4-yl)cyclo HOBT (140 mg. 0.916 mmol), EDC (176 mg, 0.916 mmol) hexyl)propanoate (320 mg, 1.162 mmol) in THF (2 mL), 30 and TEA (0.192 mL, 1.374 mmol). The mixture was stirred at RT overnight. The reaction mixture was filtered and MeOH (2 mL) and water was added LiOH (278 mg, 11.62 purified via preparative LC/MS with the following condi mmol). The mixture was heated at 70° C. for 4 h. LC-MS tions: Column: XBridge C18, 19x200 mm. 5-lum particles: indicated the completion of the reaction. The mixture was Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluo cooled to RT, neutralized with 1N HCl until pH-4, and roacetic acid; Mobile Phase B: 95:5 acetonitrile: water with extracted with EtOAc 3 times. The combined organic phases 35 0.1% trifluoroacetic acid; Gradient: 25-100% B over 20 were washed with water and brine. The solution was dried minutes, then a 4-minute hold at 100% B; Flow: 20 mL/min. over NaSO and concentrated. "H NMR (400 MHz, chlo Fractions containing the desired product were combined and roform-d) & 8.41 (d. J=5.3 Hz, 1H), 7.09-6.94 (m, 2H), dried via centrifugal evaporation to yield 4-chloro-N-(1-(4- 2.75-2.59 (m, 1H), 2.54 (d. J–3.5 Hz, 3H), 2.44 (br. S., 1H), (2-methylpyridin-4-yl)cyclohexyl)ethyl)benzamide (136.8 2.35 (t, J=7.0 Hz, 1H), 1.98-182 (m, 3H), 1.77-1.61 (m, 40 mg, 84% yield). "H NMR (500 MHz, DMSO-d) & 8.52 4H), 1.56-1.39 (m, 1H), 1.20 (d. J=6.7 Hz, 3H); MS: Anal. 8.20 (m, 2H), 7.84 (dd, J=10.6, 8.7 Hz, 2H), 7.52 (dd, J=8.4, Calc’d for CHNO. 247.16. found M+H 248.08 LC: 2.4 Hz, 2H), 7.27-6.85 (m, 2H), 4.24 (br. s. 0.5H), 3.87 (d. t=0.55 min. J=7.4 Hz, 0.5H), 3.62-3.37 (m, 1H), 2.42 (d. J=9.1 Hz, 3H), 1.94-1.31 (m, 8H), 1.20-1.02 (m, 4H). 157I. 45 1-(4-(2-Methylpyridin-4-yl)cyclohexyl)ethanamine Example 157b, c, d, e 4-Chloro-N-(1-(4-(2-methylpyridin-4-yl)cyclohexyl) 2-(4-(2-Methylpyridin-4-yl)cyclohexyl)propanoic acid ethyl)benzamide (Homochiral with Absolute and (240 mg, 0.970 mmol) (157B) was taken up in toluene (5 ml) Relative Stereochemistry not Determined) and diphenyl phosphorazidate (0.230 mL, 1.067 mmol) and 50 triethylamine (0.162 mL, 1.164 mmol) were added. The vial was sealed and heated to 70° C. After about 2 h, the reaction was cooled to rt and concentrated under reduced pressure. The crude residue was taken up in 40 mL THF and 40 mL of water and lithium hydroxide (1.589 g, 66.4 mmol) was 55 added. The reaction was stirred at rt. LCMS after 1 hour shows that the isocyanate was consumed. The reaction was acidified with 1N HCl (white precipitate forms) and extracted with EtOAc to remove DPPA related impurities. The solution was made basic with 1N NaOH (precipitate 60 forms again) and extracted with EtOAc (x5). The basic extracts were concentrated in vacuo to give 1-(4-(2-meth ylpyridin-4-yl)cyclohexyl)ethanamine (140 mg, 0.641 mmol. 66.1% yield) as a yellow oil. "H NMR (400 MHz, chloroform-d) & 8.65-8.17 (m. 1H), 7.08-6.86 (m, 2H), 2.97 65 (dd, J=8.6, 6.4 Hz, 0.5H), 2.79-2.62 (m, 1H), 2.52 (d. J=2.8 C Hz, 3H), 2.48-2.33 (m, 0.5H), 2.03-1.90 (m, 2H), 1.90-1.68
US 9,598.422 B2 134 -continued
Ex. Tr No. Name R (min)' M + HI" Stereochemistry
158c 4-chloro-N-(1-(4-(2-fluoro-3- 17.193 375.2 Homochiral with methylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined
2 N F
158d 4-chloro-N-(1-(4-(2-fluoro-3- 19497 375.2 Homochiral with methylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined
2 N F
158e 4-chloro-N-(1-(4-(2-fluoro-3- 21901 375.1 Homochiral with methylpyridin-4- absolute and yl)cycloheyxl)ethyl)benzamide relative stereochemistry N not determined)
2 N F
159a 4-chloro-N-(1-(4-(2,3- 1899-1 370.9 Diastereomer dimethylpyridin-4- Mixture yl)cyclohexyl)ethyl)benzamide N
2 N
159b 4-chloro-N-(1-(4-(2,3- 7.917-X 371.3 Homochiral with dimethylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined)
2 N
159c 4-chloro-N-(1-(4-(2,3- 8.920- 371.2 Homochiral with dimethylpyridin-4- absolute and yl)cyclohexyl)ethyl)benxamide relative stereochemistry N not determined)
2 N
159d 4-chloro-N-(1-(4-(2,3- 10.505-X 371.2 Homochiral with dimethylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined)
2 N US 9,598.422 B2 135 136 -continued
Ex. Tr No. Name R (min) M + H' Stereochemistry
159e 4-chloro-N-(1-(4-(2,3- 11.426 371.2 Homochiral with dimethylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative sterochemistry N not determined)
2 N
160a 4-chloro-N-(1-(4-(3- 1912 357.2 Diastereomer methylpyridin-4- Mixture yl)cyclohexyl)ethyl)benzamide N
2 N
4-chloro-N-(1-(4-(3- 10.662 357.3 Homochiral with methylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined)
2 N
4-chloro-N-(1-(4-(3- 13.158 357.2 Homochiral with methylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not deterermined)
2 N
160d 4-chloro-N-(1-(4-(3- 14.889 357.2 Homochiral with methylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined)
2 N
4-chloro-N-(1-(4-(3- 19.795 357.3 Homochiral with methylpyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N not determined)
2 N
161a. 4-choro-N-(1-(4-(3- 7.5424 361.2 Homochiral with fluoropyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry N41S1YY not determined) US 9,598.422 B2 137 138 -continued
Ex. Tr No. Name (min) M + H' Stereochemistry 161b 4-chloro-N-(1-(4- (3- 8.0444 361.4 Homochiral with fluoropyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry not determined)
161c 4-chloro-N-(1-(4-(3- 10.0574 361.2 Homochiral with fluoropyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative stereochemistry not determined)
161d 4-chloro-N-(1-(4-(3- 11.1774 361.3 Homochiral with fluoropyridin-4- absolute and yl)cyclohexyl)ethyl)benzamide relative sterochemistry not determined)
30 Example Né1N1twVVVVN41S1rN41N1VVVVVVV163 163B. 5-Ethoxypicolinic acid 5-Ethoxy-N-((R)-1-(cis-4-(6-fluoroquinolin-4-yl) To a solution of methyl 5-ethoxypicolinate (0.09 g, 0.497 cyclohexyl)ethyl)picolinamide 35 mmol) in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide solution (1.49 mL, 2.98 mmol). The reaction mixture was stirred at rt for 3 h. The reaction mixture was
diluted with 1 NHCl solution and ethyl acetate. The organic N-1 layer was separated and dried over MgSO. The filtrate was 40 concentrated in vacuo to give Intermediate 163B (white solid, 0.06 g., 0.359 mmol, 72.3% yield). LC-MS Anal. Calc’d for CHNO 167.06. found M+H 168.1, T=0.49 min (Method A). H NMR (400 MHz, DMSO-d) 8: 12.75 45 (br. S., 1H), 8.35 (d. J=2.6 Hz, 1H), 8.01 (d. J=8.6 Hz, 1H), 7.48 (dd, J=8.8, 2.9 HZ, 1H), 4.19 (q, J=6.9 Hz, 2H), 1.37 (t, J=6.9 Hz, 3H) Example 163 50 5-Ethoxy-N-((R)-1-(cis-4-(6-fluoroquinolin-4-yl) cyclohexyl)ethyl)picolinamide 163A. Methyl 5-ethoxypicolinate 55 To a solution of 5-ethoxypicolinic acid (14.36 mg 0.086 To a solution of methyl 5-hydroxypicolinate (0.1 g, 0.653 mmol) in DMF (1 mL) was added HATU (33 mg, 0.086 mmol) in DMF (2 mL) were added Eti (0.06 mL, 0.72 mmol). The reaction mixture was stirred at rt for 5 min, mmol), and KCO (0.135 g, 0.980 mmol). The reaction followed by addition of (R)-1-(cis-4-(6-fluoroquinolin-4-yl) mixture was stirred at rt for 2 h. The reaction mixture was cyclohexyl)ethanamine (18 mg 0.066 mmol) Intermediate diluted with saturated NaHCO solution and ethyl acetate. 60 The organic layer was separated and concentrated in vacuo 40L and N-methylmorpholine (0.032 mL, 0.264 mmol). The to give Intermediate 163A (white solid, 0.09 g, 0.497 mmol. resulting mixture was stirred at rt for 2 h. The reaction 76% yield). LC-MS Anal. Calc’d for CHNO, 181.07. mixture was concentrated in vacuo and the residue was found M+H 182.1, T, 0.66 min (Method A). H NMR dissolved in MeCH, filtered, and purified via preparative (400 MHz, methanol-d) 8: 8.29 (d. J=2.6 Hz, 1H), 8.11 (dd. 65 HPLC to give Example 163 (16 mg, 0.038 mmol, 57% J=8.6, 0.4 Hz, 1H), 7.48 (dd, J=8.7, 3.0 Hz, 1H), 4.20 (q, yield). LC-MS Anal. Calc’d for CHFNO 421.22. J=7.0 Hz, 2H), 3.94 (s, 3H), 1.45 (t, J=6.9 Hz, 3H). found M+H 422.3. T-1.63 min (Method I). H NMR (500 US 9,598.422 B2 139 140 MHz, DMSO-d) 8: 8.81 (d. J=4.4 Hz, 1H), 8.36 (d. J–9.6 -continued HZ, 1H), 8.26 (d. J=2.4 Hz, 1H), 8.07 (dd, J=9.1, 5.8 Hz, C 1H), 7.99-7.85 (m, 2H), 7.73-7.59 (m. 1H), 7.55-7.39 (m, 2H), 4.39 (d. J=6.6 Hz, 1H), 4.14 (q, J=6.9 Hz, 2H), 3.71-3.52 (m, 1H), 1.94-1.52 (m, 9H), 1.34 (t, J=6.9 Hz, 3H), 1.19 (d. J=6.4 Hz, 3H).
Example 164a, b, c, d 10 4-Chloro-N-((R)-1-(cis-4-(6-fluoroquinolin-4-yl) cyclohexyl)propyl)benzamide 4-Chloro-N-(S)-1- (cis-4-(6-fluoroquinolin-4-yl)cyclohexyl)propyl) benzamide 4-Chloro-N-((R)-1-(trans-4-(6- 15 fluoroquinolin-4-yl)cyclohexyl)propyl)benzamide 4-Chloro-N-((S)-1-(trans-4-(6-fluoroquinolin-4-yl) 164A. Ethyl cyclohexyl)propyl)benzamide (Homochiral with 2-(1,4-dioxaspiro4.5 decan-8-ylidene)acetate Absolute and Relative Stereochemistry not Deter mined) To the flask containing sodium hydride (46.1 g, 1153 mmol) was added THF (1200 mL) at 0°C. under nitrogen. Then triethyl phosphonoacetate (25.8 g., 1153 mmol) was added dropwise. The reaction mixture was stirred at 0°C. for 30 minutes. Then 1,4-dioxaspiro4.5 decan-8-one (150 g, 25 960 mmol) was added and stirred at 0° C. for 2 h. The
reaction mixture was warmed to rt and stirred for 16 h. The reaction was quenched with water (500 mL) and the mixture was concentrated in vacuo. The residue was extracted with ethyl acetate (3x1000 mL). The combined, organic layers 30 were washed with water (500 mL) and brine (500 mL) successively. The filtrate was dried over sodium sulfate and concentrated in vacuo. The crude material was purified through flash column chromatography, eluting with 0-30% ethyl acetate in petroleum ether to give Intermediate 164A (pale yellow oil, 135 g, 597 mmol, 62.1% yield). LC-MS 35 Anal. Calc’d for CHO, 226.12 found M+H. 'H NMR (400 MHz, chloroform-d) 8: 5.66 (s, 1H), 4.14 (q, J–7.2 Hz, 2H), 4.02-3.82 (m, 4H), 3.24-2.86 (m, 2H), 2.63-2.27 (m, C 2H), 1.98-1.68 (m, 4H), 1.27 (t, J–7.2 Hz, 3H). 40 164B. Ethyl 2-(1,4-dioxaspiro4.5 decan-8-yl)acetate Ethyl 2-(1,4-dioxaspiro4.5 decan-8-ylidene)acetate (13.88 g. 61.3 mmol) was taken up in EtOAc (61.3 ml) and 45 was added to a Parr hydrogenation bottle containing 10% palladium on carbon (1.306 g, 12.27 mmol) (54% w/w water) under an atmosphere of nitrogen. The reaction bottle was purged with nitrogen, then with hydrogen. After filling the bottle with hydrogen to 50 psi, the bottle was placed in 50 a Parr shaker and shaken. After 4 hours, the reaction mixture was filtered over pressed CELITER) and concentrated in vacuo to give Intermediate 164B ethyl 2-(1,4-dioxaspiro C 4.5 decan-8-yl)acetate (colorless oil, 13.78 g. 60.4 mmol. 98% yield). LC-MS Anal. Calc’d for CHO 228.14. found M+H 229.1. T, 0.83 min (Method A). "H NMR 55 (400 MHz, chloroform-d) 8: 4.31-4.08 (m, 2H), 4.00-3.86 (m, 4H), 2.22 (d. J–7.0 Hz, 2H), 1.91-1.79 (m. 1H), 1.78 1.70 (m, 4H), 1.63-1.50 (m, 2H), 1.37-1.14 (m, 5H). 164C. Ethyl 2-(4-oxocyclohexyl)acetate 60 In a 10 liter reactor was taken ethyl 2-(1,4-dioxaspiro4.5 decan-8-yl)acetate (67.5 g, 296 mmol) in acetone (5000 mL). To the reaction mixture was added 1 M HCl solution (1183 mL, 1183 mmol) and the resulting mixture was heated 65 under reflux for 2 h. The reaction mixture was concentrated to remove acetone. The residue was extracted with ethyl acetate (3x1000 mL). Combined organic layer was washed US 9,598.422 B2 141 142 with water and brine. The organic layer was dried over LC-MS Anal. Calc’d for CHFNO. 313.15. found M+H Sodium sulfate and concentrated in vacuo. The crude mate 3.14.1 T, 0.75 min (Method A). rial was purified through flash column chromatography, eluting with 0-20% ethyl acetate in petroleum ether to give 164G. Ethyl Intermediate 164C (pale yellow liquid, 40 g, 217 mmol. 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)acetate 73.4% yield). GC-MS Anal. Calc'd for CHO, 184.11 found MI 184. T-10.03 min (Method J). Intermediate 164F (4.22 g, 13.47 mmol) was dissolved in MeOH (67.3 ml) and ammonium formate (4.25 g. 67.3 164D. Ethyl 2-(4-(trifluoromethylsulfonyloxy)cy mmol) was added. The vessel was equipped with a reflux clohex-3-enyl)acetate 10 condenser and evacuated and flushed with nitrogen gas 3 times. Then palladium on carbon (0.143 g, 1.347 mmol) A 2 liter 4 neck flask was charged with 2,6-di-tert-butyl (wet, Degussa type) was added and the reaction was heated 4-methylpyridine (84.g. 407 mmol) in dichloromethane (500 to reflux for 1 hour. The reaction was cooled, concentrated mL) under nitrogen. Tf,O (55.0 mL. 326 mmol) was added in vacuo, and diluted with DCM. Solids were filtered off and dropwise. Then a solution of ethyl 2-(4-oxocyclohexyl) 15 the filtrate was concentrated to give crude Intermediate acetate (50g, 271 mmol) in dichloromethane (500 mL) was 164G (4.20 g, 13.32 mmol. 99% yield) as a mixture of cis added slowly. After completion of the addition, the reaction and trans-diastereomers. LC-MS Anal. Calcd for mixture was stirred at rt overnight. The reaction mixture was CHFNO 315.16. found M+H 316.2 T-0.76 min diluted with 1000 mL of dichloromethane and washed with (Method A). water and Sodium carbonate solution and then water. The organic layer was dried over Sodium sulfate and concen 164H. Ethyl trated in vacuo. The crude material was purified through 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)butanoate flash column chromatography, eluting with 0-10% ethyl acetate in petroleum ether to give Intermediate 164D (pale To the flask containing THF (6 mL) was added lithium yellow oil, 65 g, 206 mmol, 76% yield). GC-MS Anal. 25 diisopropylamide (2.0 M solution in THF) (3.17 mL, 6.34 Calc’d for CHFOS, 316.06 found M 317. T-10.16 mmol) at -78°C., followed by addition of 1,3-dimethyltet min (Method J). rahydropyrimidin-2(1H)-one (0.573 mL, 4.76 mmol) and a Solution of ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)ac 164E. Ethyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxa etate (1.0 g, 3.17 mmol) in THF (10 mL) dropwise at -78° borolan-2-yl)cyclohex-3-enyl)acetate 30 C. The resulting mixture turned into a brown solution and was stirred at -78° C. for 1 h, then iodoethane (0.51 mL, In 2 liter 4 neck flask was taken ethyl 2-(4-(((trifluorom 6.34 mmol) was added slowly. The reaction mixture was ethyl)sulfonyl)oxy)cyclohex-3-en-1-yl)acetate (120 g, 379 then stirred at ice bath temperature for 1 h, warmed to rt mmol), BISPIN (106 g., 417 mmol), and potassium acetate overnight. The reaction was quenched by pouring into water (112 g, 1138 mmol) in 1,4-dioxane (1200 mL) under nitro and extracting with EtOAc. The combined organic layers gen. Nitrogen was purged inside the reaction mixture for 10 35 were washed with brine, dried with MgSO filtered and minutes. Then 1,1'-bis(diphenylphosphino) ferrocene-palla concentrated in vacuo. The residue was dissolved in DCM dium dichloride dichloromethane complex (15.49 g, 18.97 and purified by silica gel flash chromatography, eluting with mmol) was added. The reaction mixture was heated at 80° 0-20% ethyl acetate in hexane to give Intermediate 164H C. for 16 h. The reaction mixture was concentrated. The (oil, 0.81 g, 2.359 mmol, 74.4% yield). LC-MS Anal. Calc’d residue was partitioned between ethyl acetate and water, 40 for CHFNO. 343.19 found M+H 344.3. T=0.87-0.88 filtered through CELITE(R) bed. The organic layer was min (Method A). H NMR (400 MHz, chloroform-d) 8: separated and the aqueous layer was extracted with ethyl 8.88-8.77 (m. 1H), 8.18-8.06 (m. 1H), 7.66 (dd, J=10.6, 2.6 acetate (3x). Combined organic layer was washed with HZ, 1H), 7.47 (ddd, J=9.2, 8.0, 2.9 Hz, 1H), 7.36 (d. J–4.6 water, brine, and dried over sodium sulfate and concentrated HZ, 1H), 4.25-4.15 (m, 2H), 3.34-3.09 (m. 1H), 2.70-2.16 in vacuo. The crude material was purified through flash 45 (m. 1H), 2.13-1.49 (m. 13H), 1.36-1.24 (m, 3H), 1.00-0.90 column chromatography, eluting with 0-10% ethyl acetate in (m, 3H). petroleum ether to give Intermediate 164E (pale yellow oil, 56 g. 190 mmol, 50.2% yield). GC-MS Anal. Calc’d for 164I. 2-(4-(6-Fluoroquinolin CHBO, 294.20 found M 295.3. T=1.10 min (Method 4-yl)cyclohexyl)butanoic acid A). H NMR (400 MHz, chloroform-d) 8: 6.52 (dd, J=4.1, 50 1.9 HZ, 1H), 4.14 (q, J=7.1 Hz, 2H), 2.62-1.97 (m, 6H), To a solution of ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclo 1.94-1.68 (m, 2H), 1.33-1.21 (m, 16H). hexyl)butanoate (0.81 g, 2.359 mmol) in THF (4 mL) and MeOH (7 mL) was added 2.0 M LiOH solution (7.1 mL, 164F. Ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohex-3- 14.2 mmol) slowly. The reaction mixture was stirred at rt en-1-yl)acetate overnight. The next day, more LiOH solution (7.1 mL, 14.2 55 mmol) was added to the reaction and the resulting mixture Ethyl 2-(4-(4.4.5.5-tetramethyl-1,3,2-dioxaborolan-2-yl) was heated at 70° C. for 28 h. The reaction mixture was cyclohex-3-en-1-yl)acetate (Intermediate 164E) (5 g, 17.00 cooled and ethyl acetate was added. The aqueous layer was mmol) was taken up in dioxane (28.3 ml) and water (7.08 separated and to the aqueous layer was added 1N HCl ml). 4-Chloro-6-fluoroquinoline (2.57 g. 14.15 mmol) was solution to adjust pH to 5-6. The resulting mixture was added followed by KCO (5.87 g. 42.5 mmol). Mixture was 60 diluted with water and CHCl: 2-propanol (2:1). The organic bubbled with nitrogen gas for 5 minutes before the addition layer was separated and dried over MgSO. The filtrate was of Pd(PhP) (0.327 g., 0.283 mmol). After the addition, the concentrated in vacuo to give Intermediate 164I as a mixture reaction was evacuated and backfilled with N three times of cis- and trans-(3:2) isomers (0.64 g, 2.029 mmol. 86% and then sealed (sealed vial parafilmed) and heated to 100° yield). LC-MS Anal. Calc’d for CHENO 315.16 found C. for 16 hours. The reaction was concentrated in vacuo and 65 M+H 316.3. T-0.72 min (Method A). "H NMR (400 purified directly via silica gel flash column chromatography MHz, chloroform-d) 8: 8.83 (d. J–4.4 Hz, 1H), 8.30-8.03 (m, to give Intermediate 164F (4.22 g, 13.47 mmol. 95% yield). 1H), 7.67 (dd, J=10.6, 2.4 Hz, 1H), 7.48 (ddd, J=9.2, 7.9, 2.6
US 9,598.422 B2 149 150 Example 197 mixture was quenched with water, THF was removed under reduced pressure. The residue was dissolved in EtOAc, 4-chloro-N-(1-(4-(pyrazolo 1.5-apyrimidin-7-yl) washed with water, brine, dried over NaSO and concen cyclohexyl)ethyl)benzamide, 4-chloro-N-((R)-1- trated. The crude was purified by ISCO (EtOAc/Hex (cis-4-(pyrazolo 1.5-alpyrimidin-7-yl)cyclohexyl) 0-30%). Fractions containing the product were concentrated ethyl)benzamide, 4-Chloro-N-((S)-1-(cis-4- to yield Intermediate 197A (1.2g, 78% yield) a light yellow (pyrazolo 1.5-apyrimidin-7-yl)cyclohexyl)ethyl) oil. "H NMR (400 MHz, CHLOROFORM-d) & 4.19 (q, benzamide, 4-chloro-N-((R)-1-(trans-4-(pyrazolo J=7.1 Hz, 2H), 4.03-3.89 (m, 4H), 2.68-2.53 (m, 2H), 1.5-alpyrimidin-7-yl)cyclohexyl)ethyl)benzamide, 2.46-2.28 (m, 2H), 1.89 (s.3H), 1.78-1.66 (m, 4H), 1.30 (t, 4-Chloro-N-((S)-1-(trans-4-(pyrazolo 1.5-apy 10 rimidin-7-yl)cyclohexyl)ethyl)benzamide Absolute J=7.1 Hz, 3H) and Relative Stereochemistry Unknown, Arbitrarily 197B. ethyl Assigned 2-(1,4-dioxaspiro4.5 decan-8-yl)propanoate
15 C C A suspension of Intermediate 143A (500 mg, 2.081 mmol) (1A) and 10% palladium on carbon (25 mg, 0.024 mmol) in H EtOAc (5 mL) was hydrogenated in a Parr shaker at 45 psi N for 6 h. The catalyst was filtered, and the filtrate was concentrated to yield Intermediate 197B (450 mg. 89% yield) as a light oil. H NMR (400 MHz, CHLOROFORM d) & 4.12 (dtt, J=10.7, 7.1, 3.6 Hz, 2H), 3.98-3.81 (m, 4H), 2.35-2.17 (m. 1H), 1.83-1.68 (m, 3H), 1.66-1.45 (m, 4H), 1.43-1.28 (m, 2H), 1.27-1.22 (m, 3H), 1.14-107 (m, 3H) 25 197C. ethyl 2-(4-oxocyclohexyl)propanoate To a solution of ethyl 2-(1,4-dioxaspiro4.5 decan-8-yl) propanoate (450 mg, 1.857 mmol) (1B) in THF (5 mL) was C C added 1M hydrogen chloride (aqueous) (0.929 mL, 3.71 30 mmol). The mixture was heated to 50° C. for 6 h. The reaction mixture was concentrated. The residue was dis solved in EtOAc, washed with water (2x), brine, dried over NaSO and concentrated. The crude was purified with ISCO (EtOAc/Hex 0-30%). Fractions containing product 35 were concentrated to yield Intermediate 197C (290 mg, 79% yield) as a clear oil. "H NMR (400 MHz, CHLOROFORM d) & 4.22-4.06 (m, 2H), 2.46-2.30 (m, 5H), 2.13-1.91 (m, 3H), 1.56-1.42 (m, 2H), 1.31-1.24 (m, 3H), 1.18 (d. J–7.1 Hz, 3H) 40 197D. ethyl 2-(4-(((trifluoromethyl)sulfonyl)oxy) cyclohex-3-en-1-yl)propanoate Intermediate 143C (200 mg, 1.01 mmol) (1C) and 2,6- 45 di-tert-butyl-4-methylpyridine (238 mg, 1.16 mmol) were dissolved in dry DCM (10 ml). To the reaction mixture trifluoromethanesulfonic anhydride (0.186 mL, 1.11 mmol) was added dropwise and stirred for 2 h. The suspension was filtered and the filtrate was diluted with DCM, washed with 50 1N HCl (2x), satd. sodium bicarb solution, water, brine and dried over NaSO and concentrated to yield Intermediate 197D (320 mg., 96% yield) as a brown oil. "H NMR (400 MHz, CHLOROFORM-d) & 5.73 (t, J=6.1 Hz, 1H), 4.28 4.05 (m, 2H), 2.52-2.17 (m, 4H), 2.08-1.79 (m, 3H), 1.49 (dt, J=11.1, 6.6 Hz, 1H), 1.31-1.20 (m, 3H), 1.19-1.04 (m, 3H) 197E. ethyl 2-(4-(4.4.5.5-tetramethyl-1,3,2-dioxa 197A. ethyl borolan-2-yl)cyclohex-3-en-1-yl)propanoate 2-(1,4-dioxaspiro4.5 decan-8-ylidene)propanoate 60 To a solution of Intermediate 143D (300 mg, 0.908 mmol) To a suspension of NaH (0.307 g., 7.68 mmol) in THF (8 (1D) in DMSO (5 mL) was added 4.4.4.4.5.5.5',5'-octam mL) cooled at 0°C. was added ethyl 2-(diethoxyphosphoryl) ethyl-2,2'-bi(1,3,2-dioxaborolane) (230 mg. 0.908 mmol) propanoate (1.830 g, 7.68 mmol) slowly. After 30 min, and potassium acetate (267 mg, 2.72 mmol). After the 1,4-dioxaspiro4.5 decan-8-one (1 g, 6.40 mmol) was 65 mixture was degassed with N for 10 min, PdCl(dppf) (19.9 added. The resulting mixture was stirred at 0°C. for 2 hours, mg, 0.027 mmol) was added. The mixture was heated to 80° then warmed up to room temperature for overnight. The C. for overnight. The mixture was partitioned between US 9,598.422 B2 151 152 EtOAc and water. The organic phase was concentrated and was stirred at rt. The reaction was acidified to pH=1 with 1N purified by ISCO. Fractions containing product were con HC1 and extracted with EtOAc to remove DPPA related centrated to yield Intermediate 197E (168 mg, 60% yield) as impurities. The organic layer was discarded. The aqueous a brown oil. "H NMR (400 MHz, CHLOROFORM-d) & layer was then basified to pH=12 with 1N NaOH and 6.66-6.40 (m. 1H), 4.31-4.00 (m, 2H), 2.34-2.26 (m. 1H), extracted with EtOAc (3x). The combined organic phases 2.25-2.19 (m. 1H), 2.19-2.04 (m, 2H), 1.95-1.75 (m, 3H), were dried with sodium sulfate, filtered, and concentrated in 1.73-1.60 (m, 1H), 1.29-124 (m, 15H), 1.13 (dd, J=11.6, 7.0 vacuo to give 197I (46.5 mg, 0.190 mmol. 63.2% yield) as Hz, 3H) an orange residue. ESI MS (M+H)"=245.2. HPLC Peak t=0.52 minutes. HPLC conditions: method A. 197F. Ethyl 2-(4-(pyrazolo 1.5-alpyrimidin-7-yl) 10 cyclohex-3-en-1-yl)propanoate Example 197a A mixture of 7-chloropyrazolo 1.5-alpyrimidine (0.193 g, (+/-)-Cis- and trans-4-chloro-N-(1-(4-(pyrazolo 1.5- 1.260 mmol), Intermediate 143E (0.400 g, 1.298 mmol), apyrimidin-7-yl)cyclohexyl)ethyl)benzamide NaCO, (0.534 g, 5.04 mmol), and Pd(PhP) (0.073 g, 15 0.063 mmol) in dioxane (11.67 ml) and water (3.89 ml) was To a solution of 197I (46.5 mg, 0.190 mmol) in THF heated at 100° C. overnight. The reaction was quenched with water and diluted with EtOAc. Layers were separated. The (1359 ul) at rt was added 4-chlorobenzoic acid (89 mg, 0.571 aqueous phase was extracted with EtOAC (3x). The organics mmol), followed by 1-(3-dimethylaminopropyl)-3-ethylcar were combined, dried over NaSO filtered, and concen bodiimide hydrochloride (109 mg, 0.571 mmol), 4-hydroxy trated to afford a brown residue. Purification of the crude benzotriazole (77 mg, 0.571 mmol) and Hunig's Base (133 material by silica gel chromatography using an ISCO ul, 0.761 mmol). The reaction was stirred at rt for 16 h. The machine (40 g column, 40 mL/min, 0-70% EtOAc in reaction was concentrated, then purified via preparative hexanes over 16 min, t, 10.5 min) gave 197F (0.224 g. LC/MS with the following conditions: Column: XBridge 0.748 mmol. 59.4% yield) as a yellow residue. ESI MS 25 C18, 19x200 mm. 5-lum particles; Mobile Phase A: 5:95 (M+H)"=300.2. HPLC Peak t—0.95 minutes. HPLC condi acetonitrile: water with 10-mMammonium acetate; Mobile tions: method A. Phase B: 95:5 acetonitrile: water with 10-mM ammonium 197G. Ethyl 2-(4-(pyrazolo 1.5-alpyrimidin-7-yl) acetate; Gradient: 20-70% B over 20 minutes, then a 5-min cyclohexyl)propanoate 30 ute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal To a solution of 143F (0.224 g., 0.748 mmol) in MeOH evaporation to afford the title compound as a mixture of 4 (3.74 ml) was added ammonium formate (0.236 g, 3.74 isomers (22.5 mg, 30%). ESI MS (M+H)"=383.0. HPLC mmol) followed by Pd/C (0.021 g, 0.202 mmol). The Peak t-1.714 minutes. Purity=98%. HPLC conditions: reaction was heated at 70° C. for 1 h. The reaction was 35 method B. filtered through CELITER) and the filter cake washed with Approximately 20.6 mg of Example 197a was resolved by CHC1. The filtrate was concentrated. The crude material the following method. The isomeric mixture was purified via was taken up in EtOAC and washed with a sat. aq. Solution of NaHCO (1x). The organic phase was dried over NaSO, preparative SFC with the following conditions: Column: filtered, and concentrated to afford 197G (220 mg, 98%) as 40 Chiral AD, 25x3 cm ID. 5-lum particles; Mobile Phase A: a yellow residue. ESI MS (M+H)"=302.2. HPLC Peak 70/30 CO/MeOH: Detector Wavelength: 220 nm: Flow: 85 t=0.94 minutes. HPLC conditions: method A. mL/min. The fractions (“Peak-1” t-7,485. “Peak-2 t=9.868, “Peak-3” t-11.635, “Peak-4” t-16.651; analyti 197H. 2-(4-(Pyrazolo 1.5-alpyrimidin-7-yl)cyclo cal conditions: Column: Chiral AD, 250x4.6 mm ID. 5-lum hexyl)propanoic acid 45 particles: Mobile Phase A: 70/30 CO/MeOH: Flow: 2.0 mL/min) were collected in MeCH. The stereoisomeric To a solution of 197G (0.1112 g, 0.369 mmol) in THF purity of each fraction was estimated to be greater than 99% (1.318 ml) and MeOH (0.527 ml) was added lithium hydrox ide (3.69 ml, 3.69 mmol). The reaction was heated at 70° C. based on the prep-SFC chromatograms. Each diasteromer or for 2.5 h, then allowed to cool to rt. The reaction was 50 enantiomer was further purified via preparative LC/MS: adjusted to pH 7 with 1N HCl, then diluted with EtOAc. Example 197b, first eluting isomer: The crude material Layers were separated. The aqueous phase was extracted was purified via preparative LC/MS with the following with EtOAc (5x). The organic phases were combined, dried conditions: Column: XBridge C18, 19x200 mm. 5-lum par over NaSO, filtered, and concentrated to afford 197H (82.7 ticles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM mg, 82%) as a yellow residue. ESI MS (M+H)"=274.1. 55 ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water HPLC Peak t—0.73 minutes. HPLC conditions: method A. with 10-mM ammonium acetate; Gradient: 20-70% B over 197I. 1-(4-(Pyrazolo 1.5-alpyrimidin-7-yl)cyclo 20 minutes, then a 5-minute hold at 100% B: Flow: 20 hexyl)ethanamine mL/min. Fractions containing the desired product were 60 combined and dried via centrifugal evaporation to afford 197H (0.0823 g, 0.301 mmol) was taken up in toluene Isomer 1 (5.0 mg., 6.6%). ESI MS (M+H)+=383.3. HPLC (1.004 ml) in a reaction vial and diphenyl phosphorazidate Peak t-1.764 minutes. Purity=96%. HPLC conditions: B. (0.071 ml, 0.331 mmol) and triethylamine (0.050 ml, 0.361 Absolute stereochemistry not determined. mmol) were added. The vial was sealed and heated to 80°C. Example 197c, second eluting isomer: The crude material After about 2 h, the reaction was cooled to rt. The crude 65 was purified via preparative LC/MS with the following residue taken up in 1 mL THF and 1 mL of water and lithium conditions: Column: XBridge C18, 19x200 mm. 5-lum par hydroxide (0.072 g, 3.01 mmol) were added. The reaction ticles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM US 9,598.422 B2 153 154 ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water 198A. (R)-3-((R)-2-((1S,4S)-4-(6-fluoroquinolin-4- with 10-mM ammonium acetate; Gradient: 20-70% B over yl)cyclohexyl)pent-4-enoyl)-4-phenyloxazolidin-2- 20 minutes, then a 5-minute hold at 100% B: Flow: 20 O mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The mate rial was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19x200 mm, 5-lum particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradi 10 ent: 35-65% B over 25 minutes, then a 2-minute hold at 65% B: Flow: 20 mL/min. Fractions containing the desired prod uct were combined and dried via centrifugal evaporation to afford Isomer 2 (5.2 mg, 7.0%). ESI MS (M+H)+=383.1. 15 HPLC Peak t-1.726 minutes. Purity=98%. HPLC condi tions: B. Absolute stereochemistry not determined. Example 197d, third eluting isomer: The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19x200 mm. 5-lum par ticles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM To a solution of Preparation 40I (50 mg 0.116 mmol) in ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water THF (2 mL) at -40°C. was added NaHMDS (1M in THF) with 10-mM ammonium acetate; Gradient: 20-70% B over (0.139 mL, 0.139 mmol) drop wise. The mixture was stirred 20 minutes, then a 5-minute hold at 100% B: Flow: 20 at -40°C. to -30°C. for 15 min. Then 3-bromoprop-1-ene 25 (28.0 mg, 0.231 mmol) in THF (0.5 mL) was added drop mL/min. Fractions containing the desired product were wise. The reaction was stirred at -20° C. for 16 h. The combined and dried via centrifugal evaporation to afford reaction was quenched at -20° C. by pouring it into satu Isomer 3 (4.7 mg, 6.3%). ESI MS (M+H)+=383.2. HPLC rated NH4C1 solution. The aqueous was extracted with Peak t—1.848 minutes. Purity=97%. HPLC conditions: B. EtOAc. The organic was washed with brine, dried over Absolute stereochemistry not determined. MgSO4, filtered and concentrated to give a crude material. 30 This crude material was added MeOH and filtered to remove Example 197e, fourth eluting isomer: The crude material the solid. The filtrate was purified with prep HPLC (Phen was purified via preparative LC/MS with the following Luna 5u 30x100 mm), 40 mL/min flow rate with gradient of conditions: Column: XBridge C18, 19x200 mm. 5-lum par 20% B-100% B Over 10 minutes Hold at 100% B for 5 min. ticles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM (A: 0.1% TFA in water/MeOH (90:10), B: 0.1% TFA in ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water 35 water/MeOH (10:90) monitoring at 254 nm. Combined with 10-mM ammonium acetate; Gradient: 20-70% B over fractions (tr-9.428 min) containing the product. After con 20 minutes, then a 5-minute hold at 100% B: Flow: 20 centration, (R)-3-((R)-2-((1s, 4S)-4-(6-fluoroquinolin-4-yl) mL/min. Fractions containing the desired product were cyclohexyl)pent-4-enoyl)-4-phenyloxazolidin-2-one (25 combined and dried via centrifugal evaporation to afford mg, 0.052 mmol. 44.8% yield) was obtained as white solid. Isomer 4 (4.5 mg, 5.9%). ESI MS (M+H)+=383.2. HPLC 40 "H NMR (400 MHz, CHLOROFORM-d) & 9.12 (d. J=5.5 Peak t—1.806 minutes. Purity=96%. HPLC conditions: B. HZ, 1H), 8.64 (dd, 5.0 Hz, 1H), 8.01-7.89 (m, 2H), 7.89-7.75 Absolute stereochemistry not determined. (m. 1H), 7.47-7.31 (m, 5H), 5.62-5.45 (m, 2H), 4.84-4.76 (m. 1H), 4.76-4.68 (m. 1H), 4.68-4.52 (m. 1H), 4.36 (dd. Example 198 45 J=9.0, 3.9 Hz, 1H), 3.55-3.33 (m, 1H), 2.49-2.35 (m, 1H), 2.33-2.21 (m, 2H), 2.12-1.97 (m, 2H), 1.93-1.65 (m, 6H) LC-MS. M+H=473.3 (tr–0.90 min) (Method A) 4-chloro-N-((R)-1-((1s.4S)-4-(6-fluoroquinolin-4- yl)cyclohexyl)but-3-en-1-yl)benzamide 198B; (R)-2-((1s.4S)-4-(6-fluoroquinolin-4-yl)cy clohexyl)pent-4-enoic acid 50
C
55
60
65 US 9,598.422 B2 155 156 To a solution of Preparation 198A (250 mg. 0.529 mmol) 0.086 mmol), followed by 4-chlorobenzoyl chloride (9.98 in THF (2 mL) at 0° C. was added 2.0 M LiOH in HO mg, 0.057 mmol). The reaction as stirred at RT for 2 h. The (0.476 mL, 0.952 mmol), followed by 30% H.O. (0.360 mL, crude material was purified via preparative LC/MS with the 3.17 mmol). The reaction was stirred at 0°C. for 10 min. following conditions: Column: XBridge C18, 19x200 mm, Then it was warmed up to RT and stirred at RT for 16 h. The 5 reaction was carefully quenched at 0° C. by addition of 5-lum particles; Mobile Phase A: 5:95 acetonitrile: water saturated Na2SO3. The pH was adjusted to 5-6 with 1N HCl with 10-mM ammonium acetate; Mobile Phase B: 95:5 and the mixture was extracted with EtOAc. The combined acetonitrile: water with 10-mM ammonium acetate; Gradi organics were dried over MgSO4, filtered and concentrated. ent: 50-100% B over 20 minutes, then a 10-minute hold at The crude material was purified with prep HPLC (9 injec 100% B; Flow: 20 mL/min. Fractions containing the desired tions) (Phen Luna 5u 30x100 mm), 40 mL/min flow rate 10 product were combined and dried via centrifugal evapora with gradient of 20% B-100% B over 10 minutes Hold at tion. The yield of the product 1 was 3.8 mg (8.70 umol. 100% B for 5 min. (A: 0.1% TFA in water/MeOH (90:10), 30.5%). B: 0.1%TFA in water/MeOH (10:90) monitoring at 254 nm. 1B (78 mg, 0.236 mmol. 44.6% yield) was obtained as white H NMR (500 MHz, DMSO-d) & 8.82 (d. J=44 Hz, 1H), solid. "H NMR (400 MHz, CHLOROFORM-d) & 9.22 (br. 15 8.27 (d. J=9.0 Hz, 1H), 8.09 (dd, J=9.0, 5.9 Hz, 1H), 7.95 (d. s., 1H), 8.63 (dd, J=9.0, 5.0 Hz, 1H), 7.98-7.75 (m, 4H), 5.85 J=10.9 Hz, 1H), 7.84 (d. J=8.3 Hz, 2H), 7.66 (t, J=7.5 Hz, (dd, J=16.9, 9.7 Hz, 1H), 5.25-5.03 (m, 2H), 3.50 (br. s. 1H), 7.57-7.43 (m, 3H), 5.90-5.73 (m. 1H), 5.07 (d. J=17.2 1H), 2.89-2.75 (m. 1H), 2.54-2.32 (m, 2H), 2.16 (d. J=10.1 HZ, 1H), 2.06 (d. J=13.2 Hz, 1H), 2.01-1.71 (m, 6H) HZ, 1H), 4.98 (d. J=10.1 Hz, 1H), 4.42 (d. J=8.9 HZ, 1H), LC-MS. M+H=328 (tr–0.69 min) (Method A) 3.39 (br. S., 1H), 2.26-2.13 (m, 1H), 1.94-1.71 (m, 7H), 1.68 (br. s. 1H), 1.62 (d. J=11.1 Hz, 1H) LC-MS: M+H=437.3 198C: (R)-1-(1S,4S)-4-(6-fluoroquinolin-4-yl)cy tr=2.23 min (Method B) clohexyl)but-3-en-1-amine
25 Example 199
N—((R)-1-((Is, 4S)-4-(6-fluoroquinolin-4-yl)cyclo 30 hexyl)but-3-en-1-yl)-1,1'-biphenyl]-4-carboxamide
35
Preparation 198B (55 mg, 0.168 mmol) taken up in toluene (1 mL) and diphenylphosphoryl azide (0.040 mL. 40 0.185 mmol) and triethylamine (0.028 mL, 0.202 mmol) was added. Vial sealed and heated to 70 C. After about 3 h, diphenylphosphoryl azide (0.040 mL, 0.185 mmol) and triethylamine (0.028 mL, 0.202 mmol) were added. The reaction was heated for another 3 h. The reaction was cooled 45 to rt and concentrated under reduced pressure. Crude residue taken up in THF (0.2 mL) and 2M LiOH (0.840 mL, 1.680 mmol). Reaction stirred at rt for 16 h. LCMS shows iso cyanate consumed. New peak with M-1 of desired at rt=0.56 min. Reaction acidified with 1 NHCl (white precipi 50 tate forms) to pH1 and extracted EtOAc to remove DPPA related impurities. The material was purified with prep HPLC (Phen Luna 5u 30x100 mm), 40 mL/min flow rate with gradient of 0% B-100% B over 10 minutes Hold at 100% B for 5 min. (A: 0.1% TFA in water/MeOH (90:10), 55 B: 0.1%TFA in water/MeOH (10:90) monitoring at 254 nm. Preparation 198C was obtained (30 mg, 0.040 mmol. Example 199 was obtained following the procedures in 23.77% yield) was obtained. LC-MS: M+H=299.2 Example 198 using 198C and 1,1'-biphenyl]-4-carbonyl (TR=0.56 min) (Method A). chloride. "H NMR (500 MHz, DMSO-d) & 8.83 (d. J–4.5 60 Example 198 HZ, 1H), 8.25 (d. J=9.2 Hz, 1H), 8.09 (dd, J=9.1, 5.8 Hz, 1H), 8.01-7.84 (m, 3H), 7.80-7.61 (m, 5H), 7.54-7.45 (m, 4-chloro-N-((R)-1-41 s, 4S)-4-(6-fluoroquinolin-4- 3H), 7.45-7.27 (m, 1H), 5.90-5.79 (m, 1H), 5.10 (d. J=17.4 yl)cyclohexyl)but-3-en-1-yl)benzamide HZ, 1H), 5.00 (d. J=9.8 Hz, 1H), 4.46 (d. J=7.9 Hz, 1H), 3.40 65 (br. s. 1H), 2.31-2.16 (m. 1H), 2.01-1.78 (m, 6H), 1.75 (br. To a solution of Preparation 198C (15 mg, 0.029 mmol) s. 2H), 1.69 (br. S., 1H), 1.63 (d. J=12.2 Hz, 1H) LC-MS: in THF (0.5 mL) at RT was added Hunig's Base (0.015 mL, M+H=470.3 tr—2.41 min (Method B) US 9,598.422 B2 157 158 Example 200 and Example 201 Ethyl 2-(4-(4.4.5.5-tetramethyl-1,3,2-dioxaborolan-2-yl) cyclohex-3-en-1-yl)acetate (5.26 g. 17.88 mmol) was taken (Chiral) N-1-(1S,4S)-4-(duinolin-3-yl)cyclohexyl) up in Dioxane (40 mL) and Water (10.00 mL). 3-bromoqui propyl)-1,1'-biphenyl]-4-carboxamide noline (3.1 g, 14.90 mmol) was added followed by potas sium carbonate (6.18 g. 44.7 mmol). Mixture was bubble with N2 for 5 minutes before addition of tetrakis(triphenyl phosphine)palladium(0) (0.344 g., 0.298 mmol). After addi tion, reaction was evacuated and backfilled with N three 10 times and then sealed and heated to 100° C. for 16 h. The Reaction was diluted with EtOAc and water. Organic was separated and washed with brine, dried over MgSO, filtered and concentrated in vacuo and purified directly via ISCO 15 (120 g column, 85 mL/min, 0-30% EtOAc in hexanes) to give Preparation 200A (4.47 g. 14.38 mmol, 96% yield). 1H NMR (400 MHz, CHLOROFORM-d) & 9.05 (d. J=2.3 Hz, 1H), 8.09 (d. J=8.4 Hz, 1H), 8.02 (d. J–2.2 Hz, 1H), 7.86-7.76 (m, 1H), 7.67 (ddd, J–8.4, 6.9, 1.5 Hz, 1H), 7.58-7.45 (m. 1H), 6.38-6.18 (m. 1H), 4.20 (q, J=7.1 Hz, 2H), 2.67-2.56 (m, 2H), 2.55-2.43 (m, 1H), 2.42-2.35 (m, 2H), 2.30-2.18 (m. 1H), 2.12-1.92 (m, 2H), 1.57 (ddt, J=12.8, 10.8, 7.9 Hz, 1H), 1.36-1.27 (m, 3H) LC-MS: 25 M+H=296.2 tr–0.74 min (Method A)
200B: ethyl 2-(4-(quinolin-3-yl)cyclohexyl)acetate 30
35
40
45 200A: ethyl 2-(4-(quinolin-3-yl)cyclohex-3-en-1-yl) acetate
50 O 1N
55 Preparation 200A (3.5 g, 11.85 mmol) was dissolved in MeOH (70 mL) and ammonium formate (3.74g, 59.2 mmol) was added. The vessel was equipped with a reflux condenser and vacated and flushed with N 3 times. Then, 10% Pd/C (1.256 g, 1.185 mmol) was added and the reaction was 60 heated at 70° C. LCMS after 1 hour shows reduction rs complete. Reaction cooled, solids were filtered off and the Na2 filtrate was concentrated to give crude material. This crude material was purified with ISCO 120g, 85 mL/min. 0-50% 65 EtOAc/Hexane. Preparation 200B (0.71 g, 2.308 mmol, 19.48% yield) was eluted with 10% EtOAc/Hexane. LC MS: M+H=302.2 tr–0.81 min (Method A) US 9,598.422 B2 159 160 200C: ethyl over MgSO, filtered and concentrated to give Preparation 2-(4-(quinolin-3-yl)cyclohexyl)butanoate 200D (400 mg, 1.076 mmol, 91% yield) as white solid. LC-MS. M+H=298.2 (tr–0.67 min) (Method A) 2OOE: 1-(4-(quinolin-3-yl)cyclohexyl)propan-1-amine 1N NH2
10
N 15 N
To a solution of Preparation 200B (920 mg, 3.09 mmol) in THF (10 mL) at 0° C. was added 1M NaHMDS in THF (7.73 mL, 7.73 mmol) drop wise. The mixture was stirred at Preparation 200D (200 mg, 0.673 mmol) taken up in 0° C. for 30 min. Then iodoethane (0.3 mL, 3.75 mmol) was toluene (2 mL) and diphenylphosphoryl azide (0.290 mL. added drop wise. The resulting mixture was stirred at 0°C. 25 1.345 mmol) and triethylamine (0.187 mL, 1.345 mmol) for 45 min. The color of the solution does not change added. Vial sealed and heated to 70 C for 16 h. The reaction much. Iodoethane (0.4 mL, 5.00 mmol) was added drop was cooled to RT and concentrated under reduced pressure. wise and the reaction was stirred at 0°C. The color of the Crude residue taken up in THF (2 mL) and 2M LiOH (2.354 solution turned to a little darker. After 1 h, iodoethane (0.15 mL, 4.71 mmol). Reaction stirred at RT for 3 days. LCMS mL, 1.875 mmol) was added and the reaction was stirred at 30 shows isocyanate consumed. New peak with M-1 of desired RT for 2 h. LC-MS shows desired product formed but still at RT=0.50 min. Reaction acidified with 1N HCl to pH1 and there is starting material left. The reaction was poured into extracted EtOAc to remove DPPA related impurities. Then a saturated NHCl solution. EtOAc was added and organic the aqueous layer was adjusted to pH 10 with 2M LiOH. was separated and washed with brine, dried over MgSO, Extracted with EtOAc (3x). Combined organics were filtered and concentrated to give a crude material. This crude washed with brine, dried over MgSO filtered and concen material was purified with ISCO 80 g column, 60 mL/min. 35 trated to give Preparation 200E (110 mg. 0.398 mmol. 0-30% EtOAc/Hexane in 40 min. The desired product was 59.1% yield) as clear liquid. LC-MS: M+H=269.2 (tr-0.50 eluted with 25% EtOAC/Hexane. Combined fractions 5-11. min) (Method A) After concentration, Preparation 200C (386 mg, 1.174 mmol. 38.0% yield) was obtained as clear liquid. LC-MS; Example 200a and Example 200b M+H=326.2 (TR=0.81, 0.82 min) (Method A) 40 N-(1-((1r,4r)-4-(duinolin-3-yl)cyclohexyl)propyl)-1, 200D: 2-(4-(quinolin-3-yl)cyclohexyl)butanoic acid 1'-biphenyl]-4-carboxamide and N-(1-((1r,4r)-4- (quinolin-3-yl)cyclohexyl)propyl)-1,1'-biphenyl-4- carboxamide (Relative and Absolute 45 Stereochemistry No Confirmed, Arbitrarily Assigned) OH
50
N 55
To a solution of Preparation 200C (385 mg, 1.183 mmol) 60 in THF (1 mL) and MeOH (5 mL) at rt was added 2M LiOH (5.91 mL, 11.83 mmol) and 1M NaOH (2.366 mL, 2.366 mmol). The reaction was stirred at 60° C. for 48 h. LC-MS still shows a little bit of starting material and methylester. Cooled to RT. The mixture was adjusted to pH 5 with 65 concentrated HC1. Extracted the aqueous layer with EtOAc. The organic was separated and washed with brine, dried
US 9,598.422 B2 165 166 -continued 0° C. and treated with potassium hexamethyldisilazide (5.50 C mL, 5.50 mmol) over 1 min. The reaction was stirred 10
min. then treated with 3,4-dichloro-2-methylpyridine (0.851 g, 5.25 mmol). The reaction was stirred 40 min. at 0°C. then quenched with aq. ammonium chloride. The phases were stirred together 1 h then extracted with 1:1 EtOAc-hexane, and the organic extract was dried and stripped to afford an oil. Prep. HPLC afforded rac-ethyl 2-((trans)-4-((3-chloro 2-methylpyridin-4-yl)oxyl)cyclohexyl)propanoate (0.47 g. 29% yield) as a golden oil. MS (ES): m/z 326 M+H". 10 t=0.78 min (Method A). 207B. rac-2-((trans)-4-((3-chloro-2-methylpyridin-4- yl)oxyl)cyclohexyl)propanoic acid 15 A solution of Preparation 207A (0.42 g, 1.289 mmol) in THF (4 mL) was treated with lithium hydroxide (0.154 g. 6.45 mmol) in water (4 mL). Methanol, ~4 mL was added to Examples 203a-d were obtained following the procedures give a single phase, and the reaction was stirred for 1 h at 50° in Examples 200 and 201 using 200E and 4-chlorobenzoyl C. The reaction was then cooled and stirred at RT. Most of chloride. The racemate was purified via preparative SFC the solvent was removed under a stream of nitrogen, and the with the following conditions: Column: Chiral IC-H 25x3 reaction was diluted to ~6 ml with water. This cloudy cm ID, 5-lum particles; Mobile Phase A: 65/35 CO2/MeOH: suspension was filtered, and the filtrate solution pH was Detector Wavelength: 220 nm: Flow: 85 mL/min. The adjusted to ~5.5 with aq. HOAc. The resulting precipitate fractions (“Peak-1 tr=5.98 min (Example 203a) and “Peak was filtered, rinsed with water, and air-dried to afford 2' tr–6.29 min (Example 203b); (“Peak-3’ tr-8.0 min rac-2-((trans)-4-((3-chloro-2-methylpyridin-4-yl)oxyl)cyclo (Example 203c) and “Peak-4 tr=9.0 min (Example 203d): 25 Examples 203a and 203b "H NMR (400 MHz, CHLO hexyl)propanoic acid (0.16 g., 42% yield) as a white solid. ROFORM-d) & 8.86 (d. J=2.2 Hz, 1H), 8.16-8.00 (m, 2H), MS (ES): m/z 298 M+H". t-0.63 min (Method A). 7.80 (d. J=8.2 Hz, 1H), 7.73-7.63 (m, 3H), 7.59-7.46 (m, 207C. rac-1-((trans)-4-((3-chloro-2-methylpyridin-4- 1H), 7.45-7.35 (m, 2H), 5.71 (d. J=10.0 Hz, 1H), 4.49-4.23 yl)oxyl)cyclohexyl)ethanamine (m. 1H), 3.06-2.83 (m, 1H), 2.24-2.05 (m. 1H), 2.00- 1.83 30 (m, 5H), 1.83-1.73 (m, 3H), 1.73-1.63 (m. 1H), 1.51-1.35 A solution of Preparation 207B (0.26g, 0.873 mmol) in (m. 1H), 1.00 (t, J=7.4 Hz, 3H) LC-MS: M+H=407.2 toluene (4.37 ml) was treated with triethylamine (0.158 ml, (tr–0.81 min) (Method A) 1.135 mmol) followed by diphenylphosphinyl azide (0.244 Examples 203c and 203d 'H NMR (400 MHz, CHLO g, 1.004 mmol). The solution was warmed to 70° C. (much ROFORM-d) & 8.86 (d. J=2.2 Hz, 1H), 8.16-8.00 (m, 2H), 35 bubbling). After 30 min., the solution was cooled and 7.80 (d. J=8.2 Hz, 1H), 7.73-7.63 (m, 3H), 7.59-7.46 (m, stripped. The residue was re-dissolved in THF (5 mL) and 1H), 7.45-7.35 (m, 2H), 5.71 (d. J=10.0 Hz, 1H), 4.49-4.23 added to a solution of lithium hydroxide (0.836 g. 34.9 (m. 1H), 3.06-2.83 (m, 1H), 2.24-2.05 (m. 1H), 2.00- 1.83 mmol) in 20 mL of water and 8 mL of THF. This mixture (m, 5H), 1.83-1.73 (m, 3H), 1.73-1.63 (m. 1H), 1.51-1.35 was stirred at RT for 30 min. then it was diluted with ether (m. 1H), 1.00 (t, J=7.4 Hz, 3H) LC-MS: M+H=407.2 40 and washed twice with 1M aq. HC1. The combined aqueous (tr–0.81 min) (Method A) phases were drained into sat. aq. Sodium carbonate (final Example 207 pH-12), and this mixture was ext, with EtOAc then 3:1 chloroform-IPA. These two organic extracts were combined, rac-4-chloro-N-(1-((trans)-4-((3-chloro-2-methyl dried, and stripped to afford rac-1-((trans)-4-((3-chloro-2- pyridin-4-yl)oxy)cyclohexyl)ethyl)benzamide 45 methylpyridin-4-yl)oxyl)cyclohexyl)ethanamine (0.18 g. 77% yield) an oil. MS (ES): m/z 269 M+H". to 0.47 min (Method A). C Example 207 50 rac-4-chloro-N-(1-((trans)-4-((3-chloro-2-methyl pyridin-4-yl)oxy)cyclohexyl)ethyl)benzamide A solution of Preparation 207C (0.01 g, 0.037 mmol) and 4-chlorobenzoic acid (6.99 mg 0.045 mmol) in DMF (0.25 55 mL) was treated with triethylamine (0.016 mL, 0.112 mmol) C followed by BOP (0.021 g, 0.048 mmol). The reaction was i stirred 2 hat RT then quenched with one drop of water and N diluted with DMF to 2 mL. This solution was then purified by prep. HPLC. Concentration of the appropriate fractions 2 N 60 afforded 0.0088 g (50%) of the title compound. MS (ES): m/z 407 M+H". to 2.05 min (Method B). 207A. rac-ethyl 2-((trans)-4-((3-chloro-2-methyl Examples 208-210 pyridin-4-yl)oxy)cyclohexyl)propanoate 65 Bop coupling (Scheme 9, below) of amine 207C (pre A Solution of ethyl rac-2-((trans)-4-hydroxycyclohexyl) pared in the preceeding example) with the appropriate propanoate (1.001 g, 5 mmol) in THF (4 mL) was cooled to benzoic acids under the conditions described for the con US 9,598.422 B2 167 168 version of 207C to Example 207 affords compounds of the TABLE 1. invention shown in Table 1 below. (All entries are racemic with trans relative stereochemistry at the cyclohexyl.)
Scheme 9 5 NH2 R
CO2H
Bop, Et3N 10 --DMF O C 5 R N i C 15 5 2 N N
2O7C R N 2
2O Exhi R (M + H)* to (min. Method B) BMTii O Example 208 F 391 1.93 BMT C 25 267222 Example 209 OMe 403 1.83 BMT O N 267223 Example 210 Me 387 1.96 BMT 2 N 30 267225 1
TABLE 2 Examples 211-225 were prepared following the procedures in Example 157 using the correspondin ridvl halide (absolute and relative stereochemistry unknown
R
O
N H
C
Example 4-chloro-N-(1-(4-(2- 2.194 411.1 Diastereomer 211 (trifluoromethyl)pyridin-4- Mixture yl)cyclohexyl)ethyl)benzamide
Example 4-chloro-N-(1-(4-(2- 9.5021C 411.3 Homochiral 212 (trifluoromethyl)pyridin-4- yl)cyclohexyl)ethyl)benzamide
US 9,598.422 B2 171 172 TABLE 2-continued
Examples 211-225 were prepared following the procedures in Example 157 using the corresponding pyridyl halide (absolute and relative stereochemistry unknown)
R
N
C
Example 4-chloro-N-(1-(4-(6- 9.3154 411.1 Homochiral 218 (trifluoromethyl)pyridin-3- yl)cyclohexyl)ethyl)benzamide
Example 4-chloro-N-(1-(4-(6- 114734D 411.1 Homochiral 219 (trifluoromethyl)pyridin-3- yl)cyclohexyl)ethyl)benzamide
Example 4-chloro-N-(1-(4-(6- 14.5454EP 411.1 Homochiral 220 (trifluoromethyl)pyridin-3- yl)cyclohexyl)ethyl)benzamide
Example N-(1-(4-(2-fluoro-3- N 2.31 Of 417.2 Diastereomer 221 methylpyridin-4- n Mixture yl)cyclohexyl)ethyl)-1,1'- biphenyl]-4-carboxamide 21
N US 9,598.422 B2 173 174 TABLE 2-continued Examples 211-225 were prepared following the procedures in Example 157 using the correspondin ridyl halide (absolute and relative stereochemistry unknown
o C Example N-(1-(4-(2-fluoro-3- 12.645-4E 417.2 Homochiral 222 methylpyridin-4- yl)cyclohexyl)ethyl)-1,1'- biphenyl]-4-carboxamide
Example N-(1-(4-(2-fluoro-3- F Co 14.189' 417.2 Homochiral 223 methylpyridin-4- yl)cyclohexyl)ethyl)-1,1'- biphenyl]-4-carboxamide Co Example N-(1-(4-(2-fluoro-3- F 15.726 417.2 Homochiral 224 methylpyridin-4- yl)cyclohexyl)ethyl)-1,1'- biphenyl]-4-carboxamide Co US 9,598.422 B2 175 176 TABLE 2-continued Examples 211-225 were prepared following the procedures in Example 157 using the correspondin ridyl halide (absolute and relative stereochemis unknown
R
O
N
C
Example N-(1-(4-(2-fluoro-3- N F 21.5654E 417.2 Homochiral 225 methylpyridin-4- N yl)cyclohexyl)ethyl)-1,1'- biphenyl]-4-carboxamide 2
O N Co Example 226 35 concentrated in vacuo to afford the title compound as an amber residue, which was used in the next step without 4-Chloro-N-((1-(6-fluoroquinolin-4-yl)-4-methylpip purification. MS(ES): m/z. 367 M+H". t-1.00 min eridin-4-yl)methyl)benzamide (Method A). 40 226B. 4-Chloro-N-(4-methylpiperidin-4-yl)methyl) benzamide To a homogeneous mixture of tert-butyl 4-(4-chloroben N C 45 Zamido)methyl)-4-methylpiperidine-1-carboxylate (226A, 0.23 mmol) in anhydrous dioxane (3 mL), under nitrogen atmosphere, was added HCl (4N in dioxane, 0.5 mL, 2.0 N mmol). The resulting mixture was stirred at ambient tem perature for 45 hours before being partitioned between water 21 50 and EtOAc. The layers were separated and the aqueous layer was extracted once more with EtOAc. The organic layers N were combined and washed with water, and this aqueous N layer was added to the original aqueous layer. The combined aqueous layer was lyophilized to afford the HCl salt of title 226A. tert-Butyl 4-((4-chlorobenzamido)methyl)-4- 55 compound as a brown residue which was used without methylpiperidine-1-carboxylate further purification. MS (ES): m/z 267 M+H". to 0.59 min (Method A). To a homogeneous mixture of tert-butyl 4-(aminom ethyl)-4-methylpiperidine-1-carboxylate (53.0 mg, 0.23 Example 226 mmol) in anhydrous DCM (2 mL), under nitrogen atmo 60 sphere, was added DIPEA (0.17 mL, 0.97 mmol) followed 4-Chloro-N-((1-(6-fluoroquinolin-4-yl)-4-methylpip by 4-chlorobenzoyl chloride (0.05 mL, 0.390 mmol). The eridin-4-yl)methyl)benzamide resulting mixture was stirred at ambient temperature for 4 hours, before being partitioned between DCM and water. To a sealable flask charged with 4-chloro-6-fluoroquino The layers were separated and the aqueous layer was 65 line (15.0 mg, 0.08 mmol) was added a homogeneous extracted twice more with DCM. These organic extracts mixture of the HCl salt of 4-chloro-N-(4-methylpiperidin were combined with the original organic layer and were 4-yl)methyl)benzamide (226B, 23.4 mg. 0.09 mmol) and US 9,598.422 B2 177 178 DIPEA (0.07 mL, 0.40 mmol) in anhydrous NMP (1 mL). Example 228 (15.6 mg: 44% yield) was prepared follow The vial was sealed and the mixture was stirred at 120° C. ing a procedure analogous to that for the synthesis of After 65 hours, the reaction mixture was cooled to room Example 227 except that 1,1'-biphenyl]-4-carbonyl chlo temperature, diluted with DMF, passed through a syringe ride was used instead of 4-chlorobenzoyl chloride, in the filter then purified via preparative HPLC/MS to afford the 5 title compound (19.4 mg: 57% yield). MS(ES): m/z 412 initial step. MS(ES): m/z 454 M+H". to 2.12 min M+H". t-191 min (Method B). H NMR (500 MHz, (Method B). H NMR (500 MHz, DMSO-d) & 8.49 (t, Hz, DMSO-d) & 8.63-8.53 (m. 2H), 8.00 (dd, J=9.1, 5.3 Hz, 1H), 8.21 (d. J=6.0 Hz, 1H), 7.90 (d. J=8.2 Hz, 2H), 1H), 7.85 (d. J=8.4 Hz, 2H), 7.81-7.71 (m, 2H), 7.52 (d. 7.79-7.65 (m, 4H), 7.48 (t, J=7.5 Hz, 2H), 7.44-7.36 (m, J=8.3 Hz, 2H), 7.10 (d. J=6.3 Hz, 1H), 3.71-3.60 (m, 1H), 1H), 7.16 (s, 1H), 7.04-6.94 (m, 1H), 3.69-3.53 (m, 2H), 3.55-3.43 (m. 1H), 3.31 (d. J=6.1 Hz, 2H), 2.95-2.85 (m, 10 3.31 (t, J=9.8 Hz, 2H), 3.25 (d. J=6.1 Hz, 2H), 1.59-148 (m, 1H), 2.56-2.54 (m. 1H), 1.79-1.68 (m, 2H), 1.58-1.49 (m, 2H), 1.41-1.32 (m, 2H), 0.99 (s.3H). 2H), 1.04 (s, 3H). Example 227 Example 229 15 4-Chloro-N-(4-methyl-1-(2-(trifluoromethyl)pyri (+/-)-4-chloro-N-(1-((1r,4r)-4-((2-(trifluoromethyl) din-4-yl)piperidin-4-yl)methyl)benzamide pyridin-4-yl)oxy)cyclohexyl)ethyl)benzamide
N C N 25 O C
21
N 30 N
Example 227 (13.9 mg; 41% yield) was prepared follow 35 Preparation 229A. ethyl 2-((1r,4r)-4-((2-(trifluorom ing a procedure analogous to that for the synthesis of ethyl)pyridin-4-yl)oxyl)cyclohexyl)propanoate Example 226 except that 4-chloro-2-(trifluoromethyl)pyri dine was used instead of 4-chloro-6-fluoroquinoline, in the final step. MS(ES): m/z 412 M+H". t1.96 min (Method B). H NMR (500 MHz, DMSO-d) & 8.57-8.48 (m, 1H), 40 8.19 (d. J=5.9 Hz, 1H), 7.78 (d. J=8.3 Hz, 2H), 7.49 (d. J=8.4 H Hz, 2H), 7.13 (s, 1H), 7.01-6.91 (m, 1H), 3.74-3.54 (m, 2H), O 3.34-3.24 (m, 2H), 3.21 (d. J=6.2 Hz, 2H), 1.55-1.43 (m, ' O 2H), 1.39-1.30 (m, 2H), 0.96 (s, 3H). O Example 228 45 N N-((4-Methyl-1-(2-(trifluoromethyl)pyridin-4-yl) piperidin-4-yl)methyl)-1,1'-biphenyl]-4-carboxam 2 ide N CF 50 To a solution of ethyl 2-((1r,4r)-4-hydroxycyclohexyl) propanoate (0.1294g, 0.646 mmol) in DMF (1.077 ml) was added NaH (0.043 g, 1.077 mmol). After 30 min, 4-bromo N 55 2-(trifluoromethyl)pyridine (0.071 ml, 0.538 mmol) as added. The reaction was heated at 80° C. overnight. Reac tion quenched with a sat. aq. Soln of NHCl and diluted with EtOAc. Layers were separated. The aqueous phase was N extracted with EtOAc (2x). The combined organic phases 60 were washed with water, dried over NaSO filtered, and 21 concentrated to afford a brown residue. Purification of the crude material by silica gel chromatography using an ISCO F N N machine (40 g column, 40 mL/min, 0-30% EtOAc in F hexanes over 14 min, t, 9.5 min) gave the title compound 65 (0.0646g, 0.187 mmol. 34.7% yield) as a colorless residue. ESI MS (M+H)+=346.2. HPLC Peak tr-1.09 minutes. HPLC conditions: A. US 9,598.422 B2 179 180 Preparation 229B. 2-((1r,4r)-4-((2-(trifluoromethyl) mmol), followed by 4-chlorobenzoyl chloride (3.38 ul, pyridin-4-yl)oxy)cyclohexyl)propanoic acid 0.026 mmol). The reaction was stirred at rt for 2 h. The crude material was purified via preparative LC/MS with the fol lowing conditions: Column: XBridge C18, 19x150 mm, 5-lum particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradi ent: 25-100% B over 20 minutes, then a 5-minute hold at OH 100% B; Flow: 20 mL/min. Fractions containing the desired 10 product were combined and dried via centrifugal evapora tion to afford the title compound (2.5 mg. 44%). ESI MS (M+H)+=427.2. HPLC Peak t-2.101 minutes. Purity=100%. HPLC conditions: B. N N CF 15 Example 230 To a solution of Preparation 229A (0.0437g, 0.127 mmol) N-(1-((1S.4s)-4-(6-(trifluoromethyl)guinolin-4-yl) in THF (0.452 ml) and MeOH (0.181 ml) was added lithium cyclohexyl)propyl)biphenyl-4-carboxamide hydroxide (1.265 ml, 1.265 mmol). The reaction was heated at 70° C. for 2 h, then allowed to cool tort. The reaction was adjusted to pH 7 with 1N HCl, then diluted with EtOAc. Layers were separated. The aqueous phase was extracted with EtOAc (3x). The organic phases were combined, dried over NaSO filtered, and concentrated to afford the title compound as a colorless residue (18.2 mg, 45% yield). ESI 25 MS (M+H)+=318.1. HPLC Peak t-0.89 minutes. HPLC conditions: A. Preparation 229C. 1-((1r,4r)-4-((2-(trifluoromethyl) pyridin-4-yl)oxy)cyclohexyl)ethanamine 30
H 35 NH2 ' O
40 21 230A. ethyl 2-(4-(6-(trifluoromethyl)cquinolin-4-yl) S. cyclohex-3-enyl)acetate N CF To a solution of 4-chloro-6-(trifluoromethyl)cquinoline Preparation 229B (18.2 mg, 0.057 mmol) taken up in 45 (2.05 g, 8.85 mmol), ethyl 2-(4-(4,4,5,5-tetramethyl-1,3,2- toluene (191 ul) and diphenyl phosphorazidate (13.59 ul, dioxaborolan-2-yl)cyclohex-3-en-1-yl)acetate (3.12 g, 0.063 mmol) and triethylamine (9.59 ul, 0.069 mmol) added. 10.62 mmol) in 1,4-dioxane (35 mL) was added potassium The vial was sealed and heated to 80°C. After about 2 h, the carbonate (3.67 g. 26.6 mmol) and water (7 mL). The reaction was cooled to rt. The reaction heated an addition 2 reaction mixture was purged with nitrogen stream for 3 min, h, then allowed to cool tort. To this reaction was added 1 mL 50 followed by addition of Pd(PhP) (0.409 g, 0.354 mmol). THF and 1 mL of water and lithium hydroxide (13.74 mg. The resulting mixture was heated at 100° C. under nitrogen 0.574 mmol). The reaction stirred at rt overnight. The stream for over night. The reaction mixture was cooled reaction was acidified to pH=1 with 1N HCl (-5.5 mL) and down and diluted with ethyl acetate and saturated NaHCO extracted with EtOAc to remove DPPA related impurities. Solution. The organic layer was separated and washed with Then, the aqueous phase was basified to pH=12 with 1N 55 NaOH and extracted with EtOAc (3x). The organic extracts sat. NaHCO solution, and dried over MgSO. The filtrate were dried with sodium sulfate, filtered, and concentrated in was concentrated in vacuo and the residue was purified via vacuo to give the title compound (3.8 mg, 0.013 mmol. silica gel flash column chromatography, eluting with 0-50% 22.98% yield) as a yellow residue. ethyl acetate in hexane to give Intermediate 230A (oil, 3.0 g, 60 8.26 mmol, 93% yield). LC-MS Anal. Calc’d for Example 229 CHFNO. 363.14. found M+H 364.5. T, 0.97 min (Method A). H NMR (400 MHz, CHLOROFORM-d) 8: (+/-)-4-chloro-N-(1-((1r,4r)-4-((2-(trifluoromethyl) 8.95 (d. J=4.5 Hz, 1H), 8.31 (s, 1H), 8.22 (d. J=8.8 Hz, 1H), pyridin-4-yl)oxy)cyclohexyl)ethyl)benzamide 7.87 (dd, J=8.8, 2.0 Hz, 1H), 7.29 (d. J=4.5 Hz, 1H), 5.86 65 (dd, J=2.8, 1.7 Hz, 1H), 4.20 (q, J=7.2 Hz, 2H), 2.65-2.24 To a solution of Preparation 229C (3.8 mg, 0.013 mmol) (m, 5H), 2.15-1.96 (m, 2H), 1.73-1.54 (m, 2H), 1.36-1.29 in THF (132 ul) at rt was added Hunig's base (6.91 ul, 0.040 (m, 3H) US 9,598.422 B2 181 182 230B. Ethyl 2-(4-(6-(trifluoromethyl)cquinolin-4-yl) mmol). The resulting mixture was heated at 65° C. over the cyclohexyl)acetate weekend. The reaction mixture was cooled down and diluted with water. To the mixture was added 1 NHCl solution to The reaction mixture of ethyl 2-(4-(6-(trifluoromethyl) adjust pH to about 5. White solid crashed out at pH 5-6. The quinolin-4-yl)cyclohex-3-en-1-yl)acetate (3.0 g, 8.26 resulting mixture was extracted with ethyl acetate twice. The mmol), ammonium formate (2.08 g. 33.0 mmol) in MeOH organic layer was separated and washed with brine, dried (50 mL) was purged with nitrogen stream for 3 min, over MgSO. The filtrate was concentrated in vacuo to give followed by addition of Pd C (0.88 g., 0.41 mmol). The Intermediate 230D as a racemate (yellow solid, 0.93 g, 2.55 resulting mixture was heated at 85°C. for 2 h. The reaction 10 mmol, 91% yield). LC-MS Anal. Calc'd for CHFNO. mixture was cooled down. The reaction mixture was filtered 365.16. found M+H 366.3. T, 0.81 min (Method A). H through a CELITER) pad and the filter cake was washed with NMR (400 MHz, DMSO-d) 8: 12.10 (br. s. 1H), 8.99 (d. MeOH. The filtrate was concentrated in vacuo. The residue J–4.6 Hz, 1H), 8.57 (s, 1H), 8.23 (d. J–8.8 Hz, 1H), 8.00 (dd. was extracted with ethyl acetate and washed with saturated NaHCO, solution, brine successively. The organic layer was 15 J=8.7, 19 Hz, 1H), 7.65 (d. J=4.6 Hz, 1H), 3.61 (d. J=10.3 dried over MgSO and the filtrate was concentrated in vacuo HZ, 1H), 1.96-1.54 (m, 11H), 1.49-1.29 (m, 1H), 0.90 (t, to give Intermediate 230B (oil, 2.6 g., 7.12 mmol. 86% yield) J=7.4 Hz, 3H) as a mixture of cis- and trans-diastereomers. LC-MS Anal. 230E 1-((1S.4s)-4-(6-(trifluoromethyl)cquinolin-4-yl) Calc’d for CHFNO, 365.16. found M+H 366.2. cyclohexyl)propan-1-amine T-0.94 min (Method A). H NMR (400 MHz, CHLORO FORM-d) 8: 9.05-8.85 (m, 1H), 8.36 (s, 1H), 8.24 (d. J=8.8 To a suspension of 2-((1S.4s)-4-(6-(trifluoromethyl)cqui HZ, 1H), 7.88 (dd, J=8.9, 1.7 Hz, 1H), 7.51-7.33 (m, 1H), nolin-4-yl)cyclohexyl)butanoic acid (0.58g, 1.587 mmol) in 4.29-4.03 (m, 2H), 3.51-3.23 (m, 1H), 2.61-2.29 (m, 2H), toluene (15 mL) were added diphenylphosphoryl azide (0.40 2.12-135 (m, 9H), 1.32-1.21 (m, 3H) 25 mL, 1.83 mmol) and triethylamine (0.24 mL, 2.06 mmol). The reaction mixture turned into clear solution after addition of TEA. The reaction mixture was heated to 70° C. for 2 h. 230C ethyl 2-((1S.4s)-4-(6-(trifluoromethyl)cquino The reaction was cooled to rt. The reaction mixture was lin-4-yl)cyclohexyl)butanoate 30 concentrated under reduced pressure. To the residue was added THF (15 mL) and 2.0 M lithium hydroxide solution (7.94 mL, 15.87 mmol) and the resulting mixture was stirred To the flask containing THF (15 mL) was added lithium at rt for 4 h. The reaction mixture was acidified with 1N HCl diisopropylamide (2.0 M solution in THF) (7.65 mL, 15.30 (white precipitate forms) and extracted with EtOAc to mmol) at -78°C., followed by addition of 1,3-dimethyltet 35 remove DPPA related impurities. Then the aqueous layer rahydropyrimidin-2(1H)-one (1.29 mL, 10.67 mmol) and a was basified with 1N NaOH (precipitate forms again) and solution of ethyl 2-(4-(6-(trifluoromethyl)cquinolin-4-yl)cy extracted with EtOAc four times. The organic extracts were clohexyl)acetate (2.6 g., 7.12 mmol) in THF (10 mL) drop combined, dried over MgSO and the filtrate was concen wise at -78° C. The resulting mixture turned into dark trated in vacuo to give light yellow oil, dried on high vacuum brown solution and stirred at -78°C. for 1 h, then iodoeth 40 over night to give Intermediate 230E (oil, 0.47 g, 1.397 ane (1.14 mL, 14.23 mmol) was added slowly. The reaction mmol, 88% yield). LC-MS Anal. Calc’d for CHFN. mixture was warmed to rt and stirred for 3 h. The reaction 336.18. found M+H 337.2. T, 0.68 min (Method A). H was quenched by pouring into water and extracted with NMR (400 MHz, CHLOROFORM-d) 8: 8.95 (d. J=4.6 Hz, EtOAc. Combined organics was washed with brine, dried 1H), 8.38 (s, 1H), 8.24 (d. J–8.8 Hz, 1H), 7.88 (dd, J=8.8, 45 1.8 Hz, 1H), 7.45 (d. J–4.6 Hz, 1H), 3.57-3.44 (m, 1H), 2.90 with MgSO, filtered and the filtrate was concentrated in (td, J–8.5, 3.0 Hz, 1H), 2.22-1.20 (m. 13H), 1.01 (d. J=15.0 vacuo. The extract was purified via silica gel flash column Hz, 3H) chromatography, eluting with 0-20% ethyl acetate in hexane to give the minor isomer and the major isomer as cis Example 230 N-(1-(1S.4s)-4-(6-(trifluoromethyl) Intermediate 230C (oil, 1.1 g, 2.77 mmol, 39% yield). 50 quinolin-4-yl)cyclohexyl)propyl)biphenyl-4-carbox LC-MS Anal. Calc’d for CHFNO,393.19. found amide M+H 394.3. T=0.97 min (Method A). H NMR (400 MHz, CHLOROFORM-d) 8: 8.97 (d. J=4.6 Hz, 1H), 8.37 (s, To a solution of 1,1'-biphenyl-4-carboxylic acid (21.2 1H), 8.24 (d. J=8.8 Hz, 1H), 7.88 (dd, J=8.8, 2.0 Hz, 1H), mg, 0.107 mmol) in DMF (1.5 mL) was added HATU (44 55 mg, 0.116 mmol). The reaction mixture was stirred at rt for 7.46 (d. J–4.6 Hz, 1H), 4.20 (q, J–7.2 Hz, 2H), 3.57-3.32 (m, 10 min, followed by addition of a solution of 1-(1S,4s)-4- 1H), 2.64 (td, J=10.8, 4.0 Hz, 1H), 2.14-1.58 (m. 11H), 1.29 (6-(trifluoromethyl)cquinolin-4-yl)cyclohexyl)propan-1- (t, J–7.2 Hz, 3H), 0.95 (t, J–74 Hz, 3H). amine (30 mg, 0.089 mmol) in THF (0.8 mL) and DIPEA (0.03 mL, 0.178 mmol). The reaction mixture was stirred at 230D. 2-((1S,4s)-4-(6-(trifluoromethyl)cquinolin-4- 60 rt for 2 h. and was concentrated in vacuo. The residue was yl)cyclohexyl)butanoic acid dissolved in MeCH, filtered, and purified via preparative HPLC to give a racemic Example 230 (33 mg 0.063 mmol. 71% yield). LC-MS Anal. Calc’d for CHFNO, 516.24. To the reaction mixture of ethyl 2-((1S.4s)-4-(6-(trifluo found M+H 517.0. T-2.02 min (Method B). H NMR romethyl)cquinolin-4-yl)cyclohexyl)butanoate (1.1 g, 2.80 65 (500 MHz, DMSO-d) 8: 9.01 (d. J=4.5 Hz, 1H), 8.55 (s, mmol) in THF (20 mL) and MeOH (8 mL) was added 1H), 8.30-8.21 (m, 1H), 8.17 (d. J=9.3 Hz, 1H), 8.03-7.91 lithium hydroxide solution (2.0 M solution) (13.98 mL, 28.0 (m, 3H), 7.79-7.67 (m, 4H), 7.61 (d. J=4.5 Hz, 1H), 7.48 (t, US 9,598.422 B2 183 184 J=7.4 Hz, 2H), 743-7.37 (m. 1H), 4.33 (d. J=8.7 Hz, 1H), -continued 4.02-3.49 (m. 1H), 1.99-1.33 (m, 11H), 0.90 (t, J–7.0 Hz, C
3H) Example 231a-e
(Absolute and Relative Stereochemistry Unknown) 4-chloro-N-(1-(4-(6-(difluoromethyl)pyridin-2-yl) cyclohexyl)propyl)benzamide 10
15 C C H N
O
25 NN 2 F
F 30
C
35 231 A. ethyl 2-(4-(6-(difluoromethyl)pyridin-2-yl) cyclohex-3-enyl)acetate To the reaction mixture of 2-bromo-6-(difluoromethyl) pyridine (1.55 g, 7.45 mmol), ethyl 2-(4-(4.4.5.5-tetram 40 ethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)acetate (2.52 g, 8.57 mmol) in 1,4-Dioxane (20 mL) was added KCO (7.45 mL, 22.36 mmol) solution and the resulting mixture was purged with nitrogen stream for 3 min, fol lowed by addition of Pd(PhP) (0.431 g, 0.373 mmol) and 45 the reaction mixture was further purged with nitrogen stream and then heated at 110° C. under nitrogen for 20 h. The reaction mixture was diluted with brine and ethyl acetate. The organic layer was separated, dried over MgSO. The filtrate was concentrated in vacuo and the residue was 50 purified via silica gel flash column chromatography, eluting with 0-20% ethyl acetate in hexane to give Intermediate C 231A (oil, 2.2 g, 7.45 mmol, 99% yield). LC-MS Anal. Calc’d for CHFNO 295.14. found M+H 296.2. T=1.10 min (Method A). H NMR (400 MHz, METHA 55 NOL-d) 8: 7.92-7.80 (m. 1H), 7.60 (dd, J=8.0, 0.8 Hz, 1H), 7.47 (d. J=7.7 Hz, 1H), 6.71 (dd, J=3.1, 2.0 Hz, 1H), 6.65-6.44 (m. 1H), 4.20-4.08 (m, 2H), 2.79-2.65 (m. 1H), 2.56-2.39 (m, 2H), 2.36 (d. J–7.0 Hz, 2H), 2.20-1.92 (m, 3H), 1.48 (dtd, J=13.0, 10.6, 5.5 Hz, 1H), 1.30-1.22 (m, 3H) 60 231B. ethyl 2-(4-(6-(difluoromethyl)pyridin-2-yl) cyclohexyl)acetate The reaction mixture of crude ethyl 2-(4-(6-(difluorom 65 ethyl)pyridin-2-yl)cyclohex-3-en-1-yl)acetate (2.1 g, 7.11 mmol), ammonium formate (1.794 g. 28.4 mmol) in MeCH (40 mL) was purged with nitrogen stream for 3 min, US 9,598.422 B2 185 186 followed by addition of 5% Pd C (0.757 g., 0.356 mmol). solid, 0.37 g, 1.22 mmol, 99% yield). LC-MS Anal. Calc’d The resulting mixture was heated at 85° C. for 2 h. The for CHFNO,297.15. found M+H 298.3, T=0.96 min reaction mixture was cooled down. The reaction mixture (Method A). was filtered and the filter cake was washed with MeOH. The H NMR (400 MHz, METHANOL-d) 8: 7.86 (td, J=7.8, filtrate was concentrated in vacuo. The residue was extracted 5 1.5 Hz, 1H), 7.50-7.37 (m, 2H), 6.86-6.47 (m, 1H), 2.99 with ethyl acetate and washed with saturated NaHCO 2.84 (m, 0.5H), 2.72 (tt, J=12.2, 3.4 Hz, 0.5H), 2.53-2.37 (m, Solution, brine Successively. The organic layer was dried 0.5H), 2.15-142 (m, 10.5H), 1.36-1.12 (m. 1H), 0.94 (td, over MgSO and the filtrate was concentrated in vacuo to J=7.4, 2.9 Hz, 3H) give Intermediate 231B (oil, 1.8 g. 6.05 mmol. 85% yield) as a mixture of cis- and trans-diastereomers. LC-MS Anal. 10 231 E. 1-(4-(6-(difluoromethyl)pyridin-2-yl)cyclo Calc’d for CHFNO, 2.97.15 found 298.2 M+H. hexyl)propan-1-amine T-109 min (Method A). "H NMR (400 MHz, CHLORO To a suspension of 2-(4-(6-(difluoromethyl)pyridin-2-yl) FORM-d) 8: 7.75 (t, J=7.8 Hz, 1H), 7.55-742 (m, 1H), cyclohexyl)butanoic acid (0.32g, 1.076 mmol) in Toluene (8 7.34-7.23 (m, 1H), 6.98 (dd, J=14.0, 7.8 Hz, 1H), 6.80-6.42 15 mL) were added diphenylphosphoryl azide (0.27 mL, 1.24 (m. 1H), 4.33-4.04 (m, 2H), 2.91-2.59 (m. 1H), 2.55-2.36 mmol) and triethylamine (0.17 mL, 1.40 mmol). The reac (m. 2H), 2.34-2.20 (m, 1H), 2.07-1.52 (m, 8H), 1.32-1.23 tion mixture in a sealed vial turned into clear solution after (m, 3H) addition of TEA. The reaction mixture was heated to 70° C. for 2.5 h. The reaction mixture was concentrated under 23 1C ethyl 2-(4-(6-(difluoromethyl)pyridin-2-yl) reduced pressure. To the residue was added THF (10 mL) cyclohexyl)butanoate and 2.0 M lithium hydroxide solution (5.4 mL, 10.76 mmol) and the resulting mixture was stirred at rt for 1 h. The To the flask containing THF (8 mL) was added lithium reaction mixture was acidified with 1N HCl (white precipi diisopropylamide (2.0 M solution in THF) (3.70 mL, 7.40 tate forms) and extracted with EtOAc to remove DPPA mmol) at -78°C., followed by addition of 1,3-dimethyltet 25 related impurities. Then the aqueous layer was basified with rahydropyrimidin-2(1H)-one (0.81 mL, 6.73 mmol) and a 1N NaOH (precipitate forms again) and extracted with solution of ethyl 2-(4-(6-(difluoromethyl)pyridin-2-yl)cy EtOAc 3 times. The basic extracts were combined, dried clohexyl)acetate (1.0 g, 3.36 mmol) in THF (10 mL) drop over MgSO and the filtrate was concentrated in vacuo to wise at -78° C. The resulting mixture turned into brown give colorless oil, dried on high vacuum over night to give solution and was stirred at -78°C. for 1 h, then iodoethane 30 Intermediate 231E (oil, 95 mg, 0.35 mmol, 33% yield). (0.54 mL, 6.73 mmol) was added slowly. The reaction LC-MS Anal. Calc'd for CHFN 268.17. found M+H mixture was then stirred at -78° C. for 0.5 h, warmed to rt 269.5. T-0.71 min (Method A). for 20 h. To the reaction mixture was added more lithium "H NMR (400 MHz, METHANOL-d) 8: 7.84 (td, J=7.8, diisopropylamide (2.0 M solution in THF) (3.70 mL, 7.40 2.2 Hz, 1H), 7.54-7.34 (m, 2H), 6.82-6.39 (m, 1H), 2.98 (dt, mmol) (1.8 mL) at ice bath temperature. The reaction 35 J=7.6, 3.5 Hz, 0.5H), 2.80-2.64 (m, 1H), 2.49 (dt, J=8.1, 4.7 mixture was stirred at ice bath temperature for 2 h. The HZ, 0.5H), 2.21-1.17 (m, 11H), 0.96 (q, J=7.4 Hz, 3H) reaction was quenched by pouring into water and extracted with EtOAc. Combined organics were washed with brine, Example 231 dried with sodium sulfate, filtered and concentrated in vacuo. The extract was purified via silica gel flash column 40 Four Isomers 4-chloro-N-(1-(4-(6-(difluoromethyl) chromatography, eluting with 0-16% ethyl acetate in hexane pyridin-2-yl)cyclohexyl)propyl)benzamide to give Intermediate 231C (oil, 0.365g, 1.122 mmol, 33% yield). LC-MS Anal. Calc'd for C.H.FNO. 325.18 To a solution of 4-chlorobenzoic acid (30.1 mg 0.192 found M+H 326.3. T-1.12 min (Method A). H NMR mmol) in DMF (1 mL) was added HATU (79 mg, 0.208 (400 MHz, CHLOROFORM-d) 8: 7.82-7.69 (m, 1H), 7.45 45 mmol). The reaction mixture was stirred at rt for 3 min, (d. J=7.5 Hz, 1H), 7.32 (d. J=7.9 Hz, 0.5H), 7.26-7.21 (m, followed by addition of a solution of Intermediate 23 1C (43 0.5H), 6.84-6.33 (m, 1H), 4.17 (qd, J=7.1, 5.9 Hz, 2H), 2.90 mg, 0.160 mmol) in THF (1 mL) and DIPEA (0.1 mL, 0.50 (dt, J=8.8, 4.4 Hz, 0.5H), 2.70 (tt, J=12.2, 3.4 Hz, 0.5H), mmol). The reaction mixture was stirred at rt for 1 h. and 2.53-2.36 (m, 0.5H), 2.18-2.09 (m, 0.5H), 2.06-1.73 (m, was concentrated in vacuo. The residue was dissolved in 5H), 1.71-1.57 (m, 4H), 1.55-1.44 (m. 1H), 1.27 (dt, J=12.8, 50 MeOH, filtered, and purified via preparative HPLC to give 7.2 Hz, 4H), 0.90 (t, J=7.4 Hz, 3H) a mixture of diastereomers Example 231a. The isomers were further separated by preparative SFC 231D. 2-(4-(6-(difluoromethyl)pyridin-2-yl)cyclo (Method R) to give first eluate Example 231b (11.3 mg, hexyl)butanoic acid 0.027 mmol. 16.8% yield). LC-MS Anal. Calc’d for 55 CHCIFNO, 406. 16. found M+H 406.9. T-2.15 min To the reaction mixture of ethyl 2-(4-(6-(difluoromethyl) (Method B). H NMR (500 MHz, DMSO-d) 8: 8.11 (d. pyridin-2-yl)cyclohexyl)butanoate (0.4 g 1.229 mmol) in J=9.1 Hz, 1H), 7.90 (t, J=7.8 Hz, 1H), 7.82 (d. J=8.3 Hz, THF (6 mL) and MeOH (3 mL) was added LiOH solution 2H), 7.57-7.41 (m, 4H), 7.02-6.68 (m, 1H), 4.02 (d. J–9.6 (6.15 mL, 18.44 mmol) at rt. The reaction mixture was then HZ, 1H), 2.86 (br. S., 1H), 2.02-1.82 (m, 2H), 1.77-1.26 (m, heated 60°C. over night. To the reaction mixture was added 60 9H), 0.82 (t, J–7.2 Hz, 3H). more THF (4 mL) and LiOH solution (6.15 mL, 18.44 Second eluate Example 231c (10.5 mg, 0.025 mmol. mmol) and the reaction mixture was heated at 60° C. for 15.8% yield). LC-MS Anal. Calc'd for CHCIFNO, another 3 days. To the reaction mixture was added 2 NHCl 406.16. found M+H 407.2. T. 2.28 min (Method B). H solution to adjust pH to about 5-6 and the resulting mixture NMR (500 MHz, DMSO-d) 8: 8.09 (d. J=9.1 Hz, 1H), 7.92 was extracted with ethyl acetate twice. The organic layer 65 (t, J=7.8 Hz, 1H), 7.84 (d. J=8.4 Hz, 2H), 7.59-7.38 (m, 4H), was separated and dried over MgSO. The filtrate was 7.04-6.73 (m, 1H), 4.03 (d. J=8.1 Hz, 1H), 2.88 (br. S., 1H), concentrated in vacuo to give Intermediate 231D (yellow 2.06-1.24 (m, 11H), 0.83 (t, J=7.2 Hz, 3H)